Nuclear translocation of the calcium-binding protein ALG-2 induced by the RNA-binding protein RBM22

By yeast two-hybrid screening using the calcium-binding protein ALG-2 as bait a new target of ALG-2 was identified, the RNA-binding protein RBM22. In order to confirm these interactions in vivo we prepared fluorescent constructs by using the monomeric red fluorescent protein to label ALG-2 and the enhanced green fluorescent protein to label RBM22. Confocal microscopy of NIH 3T3 cells transfected with either ALG-2 or RBM22 expression constructs encoding fluorescent fusion proteins alone revealed that the majority of ALG-2 was localized in the cytoplasm whereas RBM22 was located in the nucleus. When cells were co-transfected with expression vectors encoding both fusion proteins ALG-2 was found in the nucleus indicating that RBM22 which can shuttle between the cytoplasm and the nucleus may play a role in nuclear translocation of ALG-2. Using zebrafish as a model mRNA homologues of ALG-2 and RBM22 were microinjected into the blastodisc-yolk margin of zebrafish embryos at the 1-cell stage followed by monitoring the fusion proteins during development of the zebrafish. Hereby, we observed that ALG-2 alone evenly distributed within the cell, whereas in the presence of RBM22 the two proteins co-localized within the nucleus. More than 95% of the two proteins co-localized within the same area in the nucleus suggesting a functional interaction between the Ca(2+)-signaling protein ALG-2 and the RNA-binding protein RBM22.     

CCL5/CCR5 axis promotes the motility of human oral cancer cells

CCL5 (previously called RANTES) is in the CC‐chemokine family and plays a crucial role in the migration and metastasis of human cancer cells. On the other hand, the effect of CCL5 is mediated via CCR receptor. RT‐PCR and flow cytometry studies demonstrated CCR5 but not CCR1 and CCR3 mRNA in oral cancer cell lines, especially higher in those with high invasiveness (SCC4) as compared with lower levels in HSC3 cells and SCC9 cells. Stimulation of oral cancer cells with CCL5 directly increased the migration and metalloproteinase‐9 (MMP‐9) production. MMP‐9 small interfering RNA inhibited the CCL5‐induced MMP‐9 expression and thereby significantly inhibited the CCL5‐induced cell migration. Activations of phospholipase C (PLC), protein kinase Cδ (PKCδ), and NF‐κB pathways after CCL5 treatment was demonstrated, and CCL5‐induced expression of MMP‐9 and migration activity was inhibited by the specific inhibitor of PLC, PKCδ, and NF‐κB cascades. In addition, migration‐prone sublines demonstrate that cells with increasing migration ability had more expression of MMP‐9, CCL5, and CCR5. Taken together, these results indicate that CCL5/CCR5 axis enhanced migration of oral cancer cells through the increase of MMP‐9 production. J. Cell. Physiol. 220: 418–426, 2009. © 2009 Wiley‐Liss, Inc.     

Clinorotation upregulates inducible nitric oxide synthase by inhibiting AP‐1 activation in human umbilical vein endothelial cells

Alterations of nitric oxide contribute to post‐flight orthostatic intolerance. The aim of this study was to investigate the changes of inducible nitric oxide synthase (iNOS) and the mechanisms underlying regulation of iNOS by simulated microgravity in human umbilical vein endothelial cells (HUVECs). Clinorotation, a simulated‐model of microgravity, increased iNOS expression and promoter activity in HUVECs. The transactivations of NF‐κB and AP‐1 were suppressed by 24 h clinorotation. A key role for AP‐1, but not NF‐κB in the regulation of iNOS was shown. (1) PDTC, a NF‐κB inhibitor, had no effect on clinorotation upregulation of iNOS. (2) SP600125, a JNK‐specific inhibitor, which resulted in inhibition of AP‐1 activity, enhanced the iNOS expression and promoter activity in clinorotation. (3) Overexpression of AP‐1 remarkably attenuated the upregulation effect of clinorotation. These findings indicate that clinorotation upregulates iNOS in HUVECs by a mechanism dependent on suppression of AP‐1, but not NF‐κB. These results support a key role for AP‐1 in the signaling of postflight orthostatic intolerance. J. Cell. Biochem. 107: 357–363, 2009. © 2009 Wiley‐Liss, Inc.     

Biomarker discovery in asthma‐related inflammation and remodeling

Asthma is a complex inflammatory disease of airways. A network of reciprocal interactions between inflammatory cells, peptidic mediators, extracellular matrix components, and proteases is thought to be involved in the installation and maintenance of asthma‐related airway inflammation and remodeling. To date, new proteic mediators displaying significant activity in the pathophysiology of asthma are still to be unveiled. The main objective of this study was to uncover potential target proteins by using surface‐enhanced laser desorption/ionization‐time of flight‐mass spectrometry (SELDI‐TOF‐MS) on lung samples from mouse models of allergen‐induced airway inflammation and remodeling. In this model, we pointed out several protein or peptide peaks that were preferentially expressed in diseased mice as compared to controls. We report the identification of different five proteins: found inflammatory zone 1 or RELMα (FIZZ‐1), calcyclin (S100A6), clara cell secretory protein 10 (CC10), Ubiquitin, and Histone H4.     

Genetic selection system for improving recombinant membrane protein expression in E. coli

A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C‐terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I‐CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75‐fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90‐fold.     

Betel Nut Chewing Is Strongly Associated With General and Central Obesity in Chinese Male Middle‐aged Adults

Betel nut chewing has been reported to increase the risk of cardiovascular disease and all‐cause mortality. The reason is unclear. In this study, we investigated the association between betel nut chewing and general obesity (BMI ≥25 kg/m²) and central obesity (waist circumference (WC) ≥90 cm). A total of 1,049 male subjects, aged ≥40 years, were recruited from Taichung city in Taiwan in 2004. The relationships between betel nut chewing and general and central obesity were studied by multiple linear and logistic regression analyses. The prevalence of current and former betel nut chewing was 7.0 and 10.5% in our male Taiwanese cohort. Current/former betel nut chewers had a higher prevalence of general and central obesity when compared with individuals who had never chewed betel nut. Adjusted for age, diabetes, hypertension, lipids, smoking, alcohol drinking, physical activity, income, and education level, the odds ratios (ORs; 95% confidence intervals) of general and central obesity among the lower consumption of betel nut chewers were 1.78 (1.07, 2.96) and 1.19 (0.70, 2.02), respectively, compared to 2.01 (1.18, 3.41) and 1.89 (1.10, 3.23), respectively, among higher consumption chewers compared to individuals who had never chewed betel nut. The increasing ORs of general and central obesity with higher betel nut consumption revealed dose–response effects. Using multiple linear regression analyses, after adjusting for potential confounders, betel nut consumption was statistically significantly associated with BMI and WC. In conclusion, betel nut chewing was independently associated with general and central obesity in Taiwanese men. Dose–response effects of the association between betel nut consumption and general obesity as well as central obesity were found.     

Effects of brain‐derived neurotrophic factor on dopaminergic function and motor behavior during aging

Brain‐derived neurotrophic factor (BDNF) is critical in synaptic plasticity and in the survival and function of midbrain dopamine neurons. In this study, we assessed the effects of a partial genetic deletion of BDNF on motor function and dopamine (DA) neurotransmitter measures by comparing Bdnf^+/? with wildtype mice (WT) at different ages. Bdnf^+/? and WT mice had similar body weights until 12 months of age; however, at 21 months, Bdnf^+/? mice were significantly heavier than WT mice. Horizontal and vertical motor activity was reduced for Bdnf^+/? compared to WT mice, but was not influenced by age. Performance on an accelerating rotarod declined with age for both genotypes and was exacerbated for Bdnf^+/? mice. Body weight did not correlate with any of the three behavioral measures studied. Dopamine neurotransmitter markers indicated no genotypic difference in striatal tyrosine hydroxylase, DA transporter (DAT) or vesicular monoamine transporter 2 (VMAT2) immunoreactivity at any age. However, DA transport via DAT (starting at 12 months) and VMAT2 (starting at 3 months) as well as KCl‐stimulated DA release were reduced in Bdnf^+/? mice and declined with age suggesting an increasingly important role for BDNF in the release and uptake of DA with the aging process. These findings suggest that a BDNF expression deficit becomes more critical to dopaminergic dynamics and related behavioral activities with increasing age.     

Incidence of Fungal Necrotrophic and Biotrophic Pathogens in Pioneer and Shade‐tolerant Tropical Rain Forest Trees

This study assessed the hypothesis that plant life history traits determine the incidence of fungal biotrophic and necrotrophic pathogens in pioneer vs. shade‐tolerant tropical plant species. Considering that pioneer species mainly invest in induced defenses, we expected a negative relationship between the incidence of biotrophic and necrotrophic pathogens; in contrast, as shade‐tolerant species invest heavily in constitutive defenses, we expected to find no correlation between the incidence of biotrophic and necrotrophic pathogens. These ideas were evaluated by assessing standing levels of fungal damage in a set of pioneer and shade‐tolerant species from the Lacandona tropical rain forest (Mexico). The results showed that among pioneer plant species, leaves with biotrophic lesions were between 34 and 44 percent more abundant than those with necrotic lesions. In contrast, among shade‐tolerant species, the proportions of leaves with necrotic lesions were 17–23 percent higher than those of leaves with injuries caused by biotrophic pathogens. Our study suggests that tropical tree species might present different defense strategies depending on the life‐style of the pathogens that attack them, and the life history strategy of the attacked host plant species. Thus, the host constitutive and induced defenses, as well as the mechanisms used by different types of pathogens to circumvent those defenses maybe responsible for the patterns of attack observed in perennial tropical plants. Abstract in Spanish is available at http://www.blackwell‐synergy.com/loi/btp .     

Fluid shear stress‐induced JNK activity leads to actin remodeling for cell alignment

Fluid shear stress (FSS) exerted on endothelial cell (EC) surfaces induces actin cytoskeleton remodeling through mechanotransduction. This study was designed to determine whether FSS activates Jun N‐terminal kinase (JNK), to examine the spatial and temporal distribution of active JNK relative to the actin cytoskeleton in ECs exposed to different FSS conditions, and to evaluate the effects of active JNK on actin realignment. Exposure to 15 and 20 dyn/cm² FSS induced higher activity levels of JNK than the lower 2 and 4 dyn/cm² flow conditions. At the higher FSS treatments, JNK activity increased with increasing exposure time, peaking 30 min after flow onset with an eightfold activity increase compared to cells in static culture. FSS‐induced phospho‐JNK co‐localized with actin filaments at cell peripheries, as well as with stress fibers. Pharmacologically blocking JNK activity altered FSS‐induced actin structure and distribution as a response to FSS. Our results indicate that FSS‐induced actin remodeling occurs in three phases, and that JNK plays a role in at least one, suggesting that this kinase activity is involved in mechanotransduction from the apical surface to the actin cytoskeleton in ECs. J. Cell. Physiol. 226: 110–121, 2010. © 2010 Wiley‐Liss, Inc.