Insulin hypersensitivity and resistance to streptozotocin-induced diabetes in mice lacking PTEN in adipose tissue

In adipose tissue, insulin controls glucose and lipid metabolism through the intracellular mediators phosphatidylinositol 3-kinase and serine-threonine kinase AKT. Phosphatase and a tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase/AKT pathway, is hypothesized to inhibit the metabolic effects of insulin. Here we report the generation of mice lacking PTEN in adipose tissue. Loss of Pten results in improved systemic glucose tolerance and insulin sensitivity, associated with decreased fasting insulin levels, increased recruitment of the glucose transporter isoform 4 to the cell surface in adipose tissue, and decreased serum resistin levels. Mutant animals also exhibit increased insulin signaling and AMP kinase activity in the liver. Pten mutant mice are resistant to developing streptozotocin-induced diabetes. Adipose-specific Pten deletion, however, does not alter adiposity or plasma fatty acids. Our results demonstrate that in vivo PTEN is a potent negative regulator of insulin signaling and insulin sensitivity in adipose tissue. Furthermore, PTEN may be a promising target for nutritional and/or pharmacological interventions aimed at reversing insulin resistance.     

Chemoenzymatic synthesis of CD52 glycoproteins carrying native N-glycans

A facile synthesis of homogeneous CD52 glycoproteins carrying native N-glycans was achieved using an endolycosidase-catalyzed oligosaccharide transfer as the key step. The synthesis consists of two steps: the solid phase synthesis of GlcNAc-CD52 and the transfer of a high-mannose type or complex type N-glycan from Man(9)GlcNAc(2) Asn or a sialglycopeptide to the GlcNAc-CD52, under the catalysis of the endo-beta-N-acetylglucosaminidases from Arthrobacter (Endo-A) and Mucor hiemalis (Endo-M), respectively.     

Genotype-dependent expression of endothelial nitric oxide synthase (eNOS) and its regulatory proteins in cultured endothelial cells

DNA polymorphisms in endothelial nitric oxide synthase (eNOS) gene have been shown to be associated with constitutive eNOS expression and coronary artery disease (CAD). In the present study we explored the hypothesis whether genotype-dependent effects can be maintained in vitro during replication, or the effect is conditional on in vivo biological environments. Human umbilical vein endothelial cells (HUVEC) were collected and cultured from 89 normal deliveries of Mexican Americans. The cells were treated with or without cigarette smoking extracts (CSE) and genotypes of eNOS polymorphisms were determined by PCR. We measured the levels of eNOS by ELISA and its binding proteins including heat-shock protein 90 (Hsp-90) and caveolin-1 by Western blotting. The rare C allele for the promoter T786C polymorphism (0.2), and the rare 4 x 27-bp repeat allele in the intron 4 (0.30) were different from those reported in other populations. Yet, the rare T allele in the exon 7 (G894T polymorphism) was similar as others. After four passages in vitro, both the intron 4 and promoter polymorphisms maintained significant effects on eNOS mRNA levels in HUVECs (P < 0.05). However, the effects on eNOS protein and enzyme activity were less consistent. Although primary smokers had significantly lower eNOS protein levels (P < 0.05), the in vitro CSE treatment on cultured HUVECs only resulted in a significant reduction in NO levels as measured by the stable metabolites of nitrite/nitrate (P < 0.001). Neither Hsp-90 nor caveolin-1--important eNOS regulators--appears to mediate the genotypesmoking effects on eNOS expression although HUVECs did produce more Hsp-90 when exposed to CSE. Our study demonstrates that endothelial cells maintain genotype-dependent expression even after the deprivation of in vivo environment. However, the cigarette smoking-genotype interaction may require such in vivo conditions to be manifested.     

Construction of a recombinant plasmid containing multi-copy CpG motifs and its effects on the innate immune responses of aquatic animals

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides (CpG motifs) have been shown to induce potential immune responses. In this study, we designed a recombinant plasmid containing multi-copy CpG motifs, and observed its effects on innate immune responses of fish and prawn. The results showed that such plasmid DNA, compared to the vacant vector, can highly induce the activation of head kidney macrophages and the proliferation of peripheral blood leukocytes in Carassius auratus and Lateolabrax japonicus in vitro, as well as the activity of humoral defense proteins and the antibacterial activity of haemolymph in Litopenaeus vannamei in vivo. It implies that the multi-copy CpG motifs harboured in plasmid could contribute to these innate immunostimulatory effects. Therefore, the study suggested that the plasmid containing multi-copy CpG motifs might have its potential application in improving host resistance to pathogen insults in aquaculture, and have its notable advantages of high efficacy, economical cost and application to a broad range of aquatic species.     

Structural basis of diversification of fungal retinal proteins probed by site-directed mutagenesis of Leptosphaeria rhodopsin

Numerous fungal genomes encode homologs of bacteriorhodopsin (BR), but only two fungal rhodopsins were overexpressed and characterized spectroscopically. Neurospora rhodopsin (NR) is a slow-cycling sensory rhodopsin-like protein, while Leptosphaeria rhodopsin (LR) is a BR-like proton pump. Recently, we found that a conservative replacement of the cytoplasmic proton donor Asp150 by Glu converts LR into an NR-like protein. In this work, we search for structural reasons for the dramatic differences in their photochemistry by mutating the hydrogen-bonding partner of Asp150 (Thr87) and three additional residues (Thr233, Asp248, and Gly271) selected by comparison of the primary structures of NR and LR. We conclude that while these residues may contribute to the differences between LR and NR, they are not crucial for the optimization of the Schiff base reprotonation by Asp150, and that the dramatic effect of the D150E mutation is not a simple result of the introduction of a bulkier glutamate sidechain.     

Structural basis for bile salt inhibition of pancreatic phospholipase A2

Bile salt interactions with phospholipid monolayers of fat emulsions are known to regulate the actions of gastrointestinal lipolytic enzymes in order to control the uptake of dietary fat. Specifically, on the lipid/aqueous interface of fat emulsions, the anionic portions of amphipathic bile salts have been thought to interact with and activate the enzyme group-IB phospholipase A2 (PLA2) derived from the pancreas. To explore this regulatory process, we have determined the crystal structures of the complexes of pancreatic PLA2 with the naturally occurring bile salts: cholate, glycocholate, taurocholate, glycochenodeoxycholate, and taurochenodeoxycholate. The five PLA2-bile salt complexes each result in a partly occluded active site, and the resulting ligand binding displays specific hydrogen bonding interactions and extensive hydrophobic packing. The amphipathic bile salts are bound to PLA2 with their polar hydroxyl and sulfate/carboxy groups oriented away from the enzyme's hydrophobic core. The impaired catalytic and interface binding functions implied by these structures provide a basis for the previous numerous observations of a biphasic dependence of the rate of PLA2 catalyzed hydrolysis of zwitterionic glycerophospholipids in the presence of bile salts. The rising or activation phase is consistent with enhanced binding and activation of the bound PLA2 by the bile salt induced anionic charge in a zwitterionic interface. The falling or inhibitory phase can be explained by the formation of a catalytically inert stoichiometric complex between PLA2 and any bile salts in which it forms a stable complex. The model provides new insight into the regulatory role that specific PLA2-bile salt interactions are likely to play in fat metabolism.     

MMP-11 promotes papillary thyroid cell proliferation and invasion via the NF-κB pathway

Papillary thyroid carcinoma (PTC) is the most common form of thyroid cancer, and its incidence is on the rise. It has been reported that some matrix metalloproteinases (MMPs) are abnormally expressed in PTC and can be used as diagnostic markers. However, few studies have explored the underlying mechanisms by which MMPs promote tumor progression. In this study, we used microarray analysis to compare the variations of gene expression within the PTC cell populations and their adjacent normal tissues and found that MMP-11 was the most differentially expressed MMP. To investigate the role of MMP-11 in the mediation of thyroid cancer cell development, pEnter-MMP-11 plasmid, and MMP-11 small interfering RNA were applied to up- and downregulate MMP-11 expression of in cultured PTC cell lines K1 and BCPAP. The results suggested that the levels of proliferation and migration of cells transfected with MMP-11 siRNA were significantly reduced, while the levels in MMP-11-plasmid-transfected cells were increased. In terms of the mechanism, experimental data showed that the change in cyclin D1 is consistent with MMP-11 expression, which may explain the changes in proliferation. In addition, Western blot assay was conducted to analyze the p65 and activated (phospho-) p65 protein levels concomitant with MMP-11 adjustments. Variations in intracellular MMP-11 significantly altered the amount of phospho-p65 in thyroid cells, while p65 knockdown did not affect MMP-11 expression. These results suggest that MMP-11 is located upstream of p65 and regulates its activity. Interestingly, the data for the Transwell assay suggested that MMP-11 regulatory migration is also associated with the NF-κB p65 signaling pathway. In conclusion, this report describes the important role of MMP-11 in the regulation of thyroid cell proliferation and migration. Mechanistic studies have shown that cyclin D1 and p65 are important mediators in the processes, which provides a new way to study the mechanism of MMPs promoting the progression of thyroid cancer.     

Lipopolysaccharide-induced proliferation and glycolysis in airway smooth muscle cells via activation of Drp1

Abnormal airway smooth muscle cells (ASMCs) proliferation is an important pathological process in airway remodeling contributes to increased mortality in asthma. Mitochondrial dynamics and metabolism have a central role in the maintenance of the cell function. In this study, lipopolysaccharide (LPS)-induced ASMCs proliferative model was used to investigate the effect of mitochondria on the proliferation of ASMCs and the possible mechanism. We used cell and molecular biology to determine the effect of dynamin-related protein 1 (Drp1) on LPS-mediated ASMCs cell cycle progression and glycolysis. The major findings of the current study are as follows: LPS promoted an increased mitochondrial fission and phosphorylation of Drp1 at Ser616 (p-Drp1 Ser616). LPS-induced ASMCs proliferation and cell cycle progression, which was significantly inhibited application of Drp1 RNA interfering. Glycolysis inhibitor 2-deoxyglucose (2-DG) depressed ASMCs proliferative process induced by LPS stimulation. LPS caused mitochondrial metabolism disorders and aerobic glycolysis in a dependent on Drp1 activation. These results indicated that Drp1 may function as a key factor in asthma airway remodeling by mediating ASMC proliferation and cell cycle acceleration through an effect on mitochondrial metabolic disturbance.