The structural features of Trask that mediate its anti-adhesive functions

Trask/CDCP1 is a transmembrane protein with a large extracellular and small intracellular domains. The intracellular domain (ICD) undergoes tyrosine phosphorylation by Src kinases during anchorage loss and, when phosphorylated, Trask functions to inhibit cell adhesion. The extracellular domain (ECD) undergoes proteolytic cleavage by serine proteases, although the functional significance of this remains unknown. There is conflicting evidence regarding whether it functions to signal the phosphorylation of the ICD. To better define the structural determinants that mediate the anti-adhesive functions of Trask, we generated a series of deletion mutants of Trask and expressed them in tet-inducible cell models to define the structural elements involved in cell adhesion signaling. We find that the ECD is dispensable for the phosphorylation of the ICD or for the inhibition of cell adhesion. The anti-adhesive functions of Trask are entirely embodied within its ICD and are specifically due to tyrosine phosphorylation of the ICD as this function is completely lost in a phosphorylation-defective tyrosine-phenylalanine mutant. Both full length and cleaved ECDs are fully capable of phosphorylation and undergo phosphorylation during anchorage loss and cleavage is not an upstream signal for ICD phosphorylation. These data establish that the anti-adhesive functions of Trask are mediated entirely through its tyrosine phosphorylation. It remains to be defined what role, if any, the Trask ECD plays in its adhesion functions.     

Proteome profiling of Epstein-Barr virus infected nasopharyngeal carcinoma cell line: identification of potential biomarkers by comparative iTRAQ-coupled 2D LC/MS-MS analysis

Epstein-Barr virus (EBV) has been implicated in the development of nasopharyngeal carcinoma (NPC), a squamous cell carcinoma with high-occurrence in Southeast Asia and southern China. However, the underlying relationship of EBV and NPC squamous cell remains obscure. In this study, we employ a comparative iTRAQ-coupled 2D LC-MS/MS system to analyze the protein profile of NPC cell line upon EBV infection. Based on the proteome data and Western blot validation, 12 proteins were found to be significantly up-regulated and associated with signal transduction, cytoskeleton formation, metabolic pathways and DNA bindings. The interactions among NPC and EBV proteins were further analyzed and protein networks were established. Based on the functions of differentially expressed proteins, a metabolic pathway was proposed to elucidate their relationship in cytoskeleton formation, cell proliferation and apoptosis. Our results suggested a new proteome platform to analyze EBV's role in NPC squamous cell line. And these differentially expressed proteins may hold the promise as potential biomarkers for NPC diagnostics and treatment.     

Endothelin‐1 Suppresses Long‐Chain Fatty Acid Uptake and Glucose Uptake Via Distinct Mechanisms in 3T3‐L1 Adipocytes

Endothelin‐1 (ET‐1) has been demonstrated to induce insulin resistance (IR) and lipolysis, raising the possibility that ET‐1 may also contribute to the elevated fatty acid levels in IR‐associated comorbidities. We attempted to evaluate whether ET‐1 also affects the long‐chain fatty acid (LCFA) utilization in 3T3‐L1 adipocytes. The effects of chronic ET‐1 exposure on basal and insulin‐stimulated LCFA uptake, and LCFA uptake kinetics were examined in 3T3‐L1 adipocytes. Chronic exposure to ET‐1 induced IR and suppressed basal and insulin‐stimulated LCFA uptake. Given that insulin acutely stimulates LCFA uptake, there was dramatically similar trend of dose‐response curves for ET‐1‐suppressed LCFA uptake, and also similar corresponding IC₅₀ values, between basal and insulin‐stimulated states, reflecting that ET‐1 predominantly suppresses basal LCFA uptake. Results of LCFA kinetics, western blots, and CD36 inhibition using sulfosuccinimidyl oleate (SSO) revealed that suppression of LCFA uptake by ET‐1 is associated with downregulation of CD36. ET type A receptor (ET_AR) antagonist BQ‐610 reversed the IR induction and the ET‐1‐suppressed LCFA uptake. Exogenous replenishment of phosphatidylinositol (PI) 4, 5‐bisphosphate (PIP₂) prevented IR induction, but not the suppression of LCFA uptake by ET‐1. Pharmacological inhibition of the activation of mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) completely blocked the ET‐1‐suppressed LCFA uptake. Serving as an inducer of IR, ET‐1 also chronically suppresses LCFA uptake via PIP₂‐independent and ERK‐dependent pathway. The interplay between impaired glucose disposal and diminished LCFA utilization, induced by ET‐1, could worsen the dysregulation of adipose metabolism and energy homeostasis in insulin‐resistant states.     

Interactions between visitors and Formosan macaques (Macaca cyclopis) at Shou‐Shan Nature Park,Taiwan

Ecotourism involving feeding wildlife has raised public attention and is a controversial issue, especially concerning nonhuman primates. Between July 2002 and April 2005, the behavior of monkeys and tourists was collected through scan samplings, focal samplings and behavior samplings at the Shou‐Shan Nature Park located in Taiwan's second largest city—Kaohsiung. In addition, the number of tourists and monkeys was counted in different hours and places within the park. Four hundred visitors were interviewed using a questionnaire to gather data on sex, age, purpose and frequency of visit to the park. The number of tourists was significantly higher during weekends than in weekdays in all locations. Humans dominated in the initiation of interspecies interactions—the overall ratio of human‐initiated and monkey‐initiated interactions was 2.44:1. Human–monkey conflicts accounted for only 16.4% of the total interactions (n=2,166), and adult human males and adult male macaques participated in higher rates than other age/sex groups in these conflicts. Visitors showed more affiliative behavior (15.9%) than agonistic behavior (8%) toward the macaques. In response to visitors' threat or attack, the Formosan macaques mostly showed submissive behavior with bared teeth, squealed or ran away to avoid confrontation (69.1%)—only few responded with counteraggression (18.7%). This study for the first time provided evidence that food provisioning increased both the frequency and duration of aggression among Formosan macaques (P<0.001). During food provisioning, the average frequency and the duration of agonistic events of macaques were more than 4 times higher compared with those without food provisioning. The average frequency of food provision by tourists was 0.73 times/hr—more than twice the incident that monkeys grabbed the food from tourists (0.34 times/hr). If people refrain from feeding monkeys and destroying the city park's natural vegetation, monkeys can be used to educate public about nature conservation in an urban setting. Am. J. Primatol. 71:214–222, 2009. © 2008 Wiley‐Liss, Inc.     

Effect of environmental factors on the production of oxygenated unsaturated fatty acids from linoleic acids by Bacillus megaterium ALA2

We identified [Hou CT (2003) New uses of vegetable oils: novel oxygenated fatty acids by biotransformation. SIM News 53:56–61] many novel oxygenated fatty acids produced from linoleic acid by Bacillus megaterium ALA2: 12,13,17-trihydroxy-9(Z)-octadecenoic acid (12,13,17-THOA); 12,13,16-trihydroxy-9(Z)-octadecenoic acid (12,13,16-THOA); 12-hydroxy-13,16-epoxy-9(Z)-octadecenoic acid; and 12,17;13,17-diepoxy-16-hydroxy-9(Z)-octadecenoic acid. 12,13,17-THOA, the main product, has antiplant pathogenic fungal activity. To develop an industrial process for the production of these new oxygenated fatty acids by strain ALA2, the effect of environmental factors on the production and their impact on the amount of various products were studied. Dextrose at 5 g/l was the optimum amount for the carbon source. A combination of 15 g yeast extract and 10 g tryptone showed good results as nitrogen sources. Among the metal ions tested, the optimum concentrations for the reaction for the different ions were as follows (in mM): magnesium 2.0, iron 0.5, zinc 0.1, nickel 0.01, and cobalt 0.05. Copper ions did not affect the production of oxygenated products; however, manganese ions inhibited the reaction. Addition of these metal ions did not alter the distribution of products. The optimum temperature and pH for the production of THOAs were 30°C and pH 6.5. Time course studies showed 40–48 h is the optimum for the production of both THOAs. These data provide the basis for engineering scale-up production of these new products.     

Insulin-like growth factor-1 increases endothelin receptor A levels and action in cultured rat aortic smooth muscle cells

Insulin is known to cause an increase in endothelin-1 (ET-1) receptors in vascular smooth muscle cells (SMCs), but the effect of insulin-like growth factor 1 (IGF-1) on ET-1 receptor expression is not known. We therefore carried out the present study to determine the effect of IGF-1 on the binding of ET-1 to, and ET type A receptor (ETAR) expression and ET-1-induced 3H-thymidine incorporation in, vascular SMCs. In serum-free medium, IGF-1 treatment increased the binding of 125I-ET-1 to SMC cell surface ET receptors from a specific binding of 20.1%+/-3.1% per mg of protein in control cells to 45.1%+/-8.6% per mg of protein in cells treated with IGF-1 (10 nM). The effect of IGF-1 was dose-related, with a significant effect (1.4-fold) being seen at 1 nM. The minimal time for IGF-1 treatment to be effective was 30 min and the maximal effect was reached at 6 h. Immunoblotting analysis showed that ETAR expression in IGF-1-treated cells was increased by 1.7-fold compared to controls. Levels of ETAR mRNA measured by the RT-PCR method and Northern blotting were also increased by 2-fold in IGF-1-treated SMCs. These effects of IGF-1 were abolished by cycloheximide or genistein. Finally, ET-1-stimulated thymidine uptake and cell proliferation were enhanced by IGF-1 treatment, with a maximal increase of 3.2-fold compared to controls. In conclusion, in vascular SMCs, IGF-1 increases the expression of the ET-1 receptor in a dose- and time-related manner. This effect is associated with increased thymidine uptake and involves tyrosine kinase activation and new protein synthesis. These findings support the role of IGF-1 in the development of atherosclerotic, hypertensive, and diabetic vascular complications.     

Profound difference of metabolic pharmacokinetics between pure glycyrrhizin and glycyrrhizin in licorice decoction

To investigate the difference of metabolic pharmacokinetics between pure glycyrrhizin (GZ) and GZ in licorice decoction, six New Zealand White rabbits were orally given pure GZ and licorice decoction containing equivalent content of GZ in a randomized crossover design. HPLC methods were used for the quantitation of GZ and glycyrrhetic acid (GA) in serum. The results indicated that the areas under curves (AUCs) of GZ and GA after administration of licorice decoction were significantly higher than those after pure GZ. This result was contradictory with that obtained in rats. To explore the mechanism of the pharmacokinetic difference, feces of rabbits, rats, pigs and humans were used to investigate the presystemic metabolism of pure GZ and GZ in licorice decoction. The results indicated that pure GZ was hydrolyzed to GA more rapidly and to a greater extent than that in licorice decoction by various feces. In addition, when pure GZ was fermented, the metabolic profiles of GA and 3-dehydroGA in rabbit feces were quite different from other feces. In conclusion, the bioavailabilities of GZ and GA are significantly better from licorice than from pure GZ in rabbits but the presystemic metabolism of pure GZ in rabbit is rather different from that in rat, pig and human.     

Preparation of recombinant thioredoxin fused N-terminal proCNP: Analysis of enterokinase cleavage products reveals new enterokinase cleavage sites

C-type natriuretic peptide (CNP) acts as a paracrine hormone to dilate blood vessels and is also required for the growth of long bones. In vivo, CNP is produced by cleavage from the C-terminal end of a larger proCNP peptide. The remaining N-terminal proCNP fragment (NT-proCNP) escapes into the circulation where its concentration is much higher than that of CNP due presumably to a lower clearance rate. Our strategy to obtain large quantities of pure NT-proCNP for further physiological investigations was to express it as a fusion protein with His(6)-tagged thioredoxin followed by cleavage using enterokinase to yield NT-proCNP alone. We have successfully designed and artificially synthesized the coding sequence specifying both mouse and human NT-proCNP with built-in codon bias towards Escherichia coli codon preference. An enterokinase recognition sequence was incorporated immediately upstream of the NT-proCNP coding sequence to allow the fusion protein to be cleaved without leaving any extra residues on the NT-proCNP peptide. High levels of fusion proteins were obtained, constituting 50-58% of total bacterial proteins. Greater than 90% of recombinant thioredoxin/NT-proCNP was expressed in the soluble form and purified to near homogeneity in a single chromatographic step using nickel as the metal ion in IMAC. A time course analysis of the products released from enterokinase cleavage of the recombinant proteins by ESI-MS revealed three sensitive secondary cleavage sites: two were located on vector-associated sequences linking the thioredoxin moiety and NT-proCNP, and one at the C-terminal end of NT-proCNP. Clearly, substrate specificity of both the native and recombinant forms of enterokinase for the recognition sequence DDDDK was by no means exclusive. Hydrolysis at the unexpected LKGDR site located towards the carboxyl end on NT-proCNP was significantly more efficient than at the internally sited DDDDK target sequence. However, when this same sequence was sited internally replacing the DDDDK in another construct of thioredoxin/mouse NT-proCNP, it was found to be poorly processed by enterokinase. Our results showed that non-target sequences can be preferentially recognized over the canonical DDDDK sequence when located accessibly at the ends of proteins.     

Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel accretion

Regulation of gap junction (GJ) organization is critical for proper function of excitable tissues such as heart and brain, yet mechanisms that govern the dynamic patterning of GJs remain poorly defined. Here, we show that zonula occludens (ZO)-1 localizes preferentially to the periphery of connexin43 (Cx43) GJ plaques. Blockade of the PDS95/dlg/ZO-1 (PDZ)-mediated interaction between ZO-1 and Cx43, by genetic tagging of Cx43 or by a membrane-permeable peptide inhibitor that contains the Cx43 PDZ-binding domain, led to a reduction of peripherally associated ZO-1 accompanied by a significant increase in plaque size. Biochemical data indicate that the size increase was due to unregulated accumulation of gap junctional channels from nonjunctional pools, rather than to increased protein expression or decreased turnover. Coexpression of native Cx43 fully rescued the aberrant tagged-connexin phenotype, but only if channels were composed predominately of untagged connexin. Confocal image analysis revealed that, subsequent to GJ nucleation, ZO-1 association with Cx43 GJs is independent of plaque size. We propose that ZO-1 controls the rate of Cx43 channel accretion at GJ peripheries, which, in conjunction with the rate of GJ turnover, regulates GJ size and distribution.