SPLUNC1 Regulates Cell Progression and Apoptosis through the miR-141-PTEN/p27 Pathway,but Is Hindered by LMP1

Little is known about the role of the host defensive protein short palate, lung and nasal epithelium clone 1 (SPLUNC1) in the carcinogenesis of nasopharyngeal carcinoma (NPC). Here we report that SPLUNC1 plays a role at a very early stage of NPC carcinogenesis. SPLUNC1 regulates NPC cell proliferation, differentiation and apoptosis through miR-141, which in turn regulates PTEN and p27 expression. This signaling axis is negatively regulated by the EBV-coded gene LMP1. Therefore we propose that SPLUNC1 suppresses NPC tumor formation and its inhibition by LMP1 provides a route for NPC tumorigenesis.     

Inhibition of retinal neovascularization by gene transfer of small interfering RNA targeting HIF-1alpha and VEGF

Retinal neovascularization (NV) occurs in various ocular disorders including proliferative diabetic retinopathy, retinopathy of prematurity and secondary neovascular glaucoma, which often result in blindness. Vascular endothelial growth factor (VEGF) is an essential growth factor for angiogenesis, and is particularly regulated by hypoxia inducible factor-1alpha (HIF-1alpha) under hypoxic conditions. Therefore, HIF-1alpha and VEGF could provide targets for therapeutic intervention on retinal NV. In this study, we investigated the inhibitory effects of small interfering RNA (siRNA) targeting HIF-1alpha and VEGF on the expression of HIF-1alpha and VEGF in human umbilical vein endothelial cells (HUVEC) in vitro and on retinal NV in vivo. siRNA-expressing plasmids targeting human HIF-1alpha (HIF-1alpha siRNA) and human VEGF(165) (VEGF siRNA) were constructed. They were transfected and co-transfected to HUVEC and C57BL/6J mice of ischemic retinopathy model. HIF-1alpha siRNA and VEGF siRNA specifically downregulated HIF-1alpha and VEGF at both mRNA and protein levels in vitro and in vivo. Neovascular tufts and neovascular nuclei were decreased in gene therapy group compared to control hypoxia group. Co-transfection of HIF-1alpha siRNA and VEGF siRNA resulted in maximal effects on VEGF suppression in vitro and in vivo. It also manifested the maximal inhibitory effect on retinal NV. These results indicate that the application of HIF-1alpha siRNA and VEGF siRNA technology holds great potential as a novel therapeutic for retinal NV.     

Genomic analysis of allelopathic response to low nitrogen and barnyardgrass competition in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To explore the molecular mechanism of allelopathic rice in response to low nitrogen (N) supply or accompanying weed stress, allelopathic rice PI 312777 and its counterpart Lemont were grown under low N supply or co-cultured with barnyardgrass [Echinochloa crus-galli (L.) Beauv.] in hydroponics. The suppression subtractive hybridization (SSH) technique was employed to isolate the up-regulated genes in the treated rice accession. The results indicated that the expression of the genes associated with N utilization was significantly up-regulated in allelopathic rice PI 312777, and the higher efficiency of N uptake and its utilization were also detected in PI 312777 than that in Lemont when the two rice accessions were exposed to low N supply. This result suggested that the allelopathic rice had higher ability to adapt to low N stress than its non-allelopathic counterpart. However, a different response was observed when the allelopathic rice was exposed to accompanying weed (barnyardgrass) co-cultured in full Hoagland solution (normal N supply). It showed that the expression of the genes associated with allelochemical synthesis and its detoxification were all up-regulated in the allelopathic rice when co-cultured with the target weed under normal N supply. The results suggested that the allelopathic rice should be a better competitor in the rice-weed co-culture system, which could be attributed to increasing de novo biosynthesis and detoxification of allelochemicals in rice, consequently resulting in enhanced allelopathic effect on the target and preventing the autotoxicity in this process. These findings suggested that the accompanying weed, barnyardgrass is not only the stressful factor, but also one of the triggers in activating allelopathy in rice. This implies that the allelopathic rice is sensible of the existing target in chemical communication.     

A novel compound derived from danshensu inhibits apoptosis via upregulation of heme oxygenase-1 expression in SH-SY5Y cells

Background

Heme oxygenase-1 (HO-1) has potential anti-apoptotic properties. A novel compound [4-(2-acetoxy-3-((R)-3-(benzylthio)-1-methoxy-1-oxopropan-2- ylamino)-3-oxopropyl)-1,2-phenylene diacetate (DSC)] was synthesized by joining danshensu and cysteine through an appropriate linker. This study investigated if the cytoprotective properties of DSC involved the induction of HO-1.

Methods

We evaluated the cytoprotective effects of DSC on H₂O₂-induced cell damage, apoptosis, intracellular and mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨ_m) loss, and apoptosis-related proteins expression and its underlying mechanisms.

Results

DSC concentration-dependently attenuated cell death, lactate dehydrogenase release, intracellular and mitochondrial ROS production, and ΔΨ_m collapse, modulated apoptosis-related proteins (Bcl-2, Bax, caspase-3, p53, and cleaved PARP) expression, and inhibited phosphorylation of extracellular signal-regulated kinase 1/2 in SH-SY5Y cells induced by H₂O₂. In addition, DSC concentration-dependently induced HO-1 expression associated with nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf-2), while the effect of DSC was inhibited by a phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Furthermore, the protective effect of DSC on H₂O₂-induced cell death was abolished by HO-1 inhibitor ZnPP, but was mimicked by carbon monoxide-releasing moiety CORM-3 or HO-1 by-product bilirubin. Finally, DSC inhibited H₂O₂-induced changes of Bcl-2, Bax, and caspase-3 expression, and all of these effects were reversed by HO-1 silencing.

Conclusions

Induction of HO-1 may be, at least in part, responsible for the anti-apoptotic property of DSC, an effect that involved the activation of PI3K/Akt/Nrf-2 axis.

General significance

DSC might have the potential for beneficial therapeutic interventions for neurodegenerative diseases.     

Insulin-stimulated Interaction between insulin receptor substrate 1 and p85alpha and activation of protein kinase B/Akt require Rab5

Binding of insulin to the insulin receptor initiates a cascade of protein phosphorylation and effector recruitment events leading to the activation of multiple distinct signaling pathways. Previous studies suggested that the diversity and specificity of insulin signal transduction are accomplished by both subcellular localization of receptor and the selective activation of downstream signaling molecules. The small GTPase Rab5 is a key regulator of endocytosis. Three Rab5 isoforms (Rab5a, -5b, and -5c) have been identified. Here we exploited the RNA interference technique to specifically knock down individual Rab5 isoforms to determine the cellular function of Rab5 in distinct insulin signaling pathways. Small interference RNA against a single Rab5 isoform had no effect on protein kinase B (PKB)/Akt or MAPK activation by insulin in NIH3T3 cells overexpressing human insulin receptor. However, simultaneous knockdown of all three Rab5 isoforms dramatically attenuated PKB/Akt activation by insulin without affecting MAPK activation. This inhibition of PKB/Akt activation was because of the impaired interaction between insulin receptor substrate 1 and the p85alpha subunit of phosphatidylinositol 3-kinase. These results indicate a requirement of Rab5 in presenting p85 to insulin receptor substrate 1. Additional evidence supporting a role for Rab5 was suggested by studies with GAPex-5, a vps9 domain containing exchange factor. Down-regulation of GAPex-5 impaired insulin-stimulated PKB/Akt activation. Collectively, this study indicates the involvement of Rab5 in insulin signaling.     

The influence of hydrological regimes on sex ratios and spatial segregation of the sexes in two dioecious riparian shrub species in northern Sweden

River management practices have altered the hydrological regimes of many rivers and also altered the availability of regeneration niches for riparian species. We investigated the impact of changed hydrological regimes on the sex ratios and the Spatial Segregation of the Sexes (SSS) in the dioecious species Salix myrsinifolia Salisb.–phylicifolia L. and S. lapponum L. by studying the free-flowing Vindel River and the regulated Ume River in northern Sweden. We surveyed sex ratios of these species in 12 river reaches on the Vindel River and in 17 reaches on the Ume River. In addition, we surveyed the sex and location above mean river stage of 1,002 individuals across both river systems to investigate the SSS of both species. Cuttings were collected from male and female individuals of S. myrsinifolia–phylicifolia from both rivers and subjected to four different water table regimes in a greenhouse experiment to investigate growth response between the sexes. We found an M/F sex ratio in both river systems similar to the regional norm of 0.62 for S. myrsinifolia–phylicifolia and of 0.42 for S. lapponum. We found no evidence of SSS in either the free-flowing Vindel River or the regulated Ume River. In the greenhouse experiment, hydrological regime had a significant effect on shoot and root dry weight and on root length. Significantly higher shoot dry weights were found in females than in males and significantly different shoot and root dry weights were found between cuttings taken from the two rivers. We concluded that changed hydrological regimes are likely to alter dimensions of the regeneration niche and therefore to influence sex ratios and SSS at an early successional stage, making it difficult to find clear spatial patterns once these species reach maturity and can be sexed.     

Phylogeny of the East Asian botiine loaches (Cypriniformes,Botiidae) inferred from mitochondrial cytochrome <Emphasis Type="Italic">b</Emphasis> gene sequences

The Botiinae have traditionally represented a subfamily of the Cobitidae. At present, the classification and phylogenetic relationships of the Botiinae are controversial. To address systematic and phylogenetic questions concerning this group, we sequenced the complete cytochrome b gene from 34 samples, of which 24 represented 13 species of the East Asian botiine fishes, while the other 10 were non-botiine loach species. For the 1140 bp sequences determined, 494 sites were variable ones, of which 424 were parsimony informative. With Myxocyprinus asiaticus as an outgroup, molecular phylogenetic trees were constructed using the neighbor-joining, maximum parsimony, maximum likelihood and Bayesian methods. All molecular phylogenetic trees revealed that botiine fishes form a monophyletic group and are distantly related to other loaches, suggesting that the Botiinae should be placed in their own family. Within the Botiinae, there are three genera; Botia, Parabotia, andLeptobotia, each genus forming a monophyletic group, with the genus Botia as the most ancestral split. Our molecular results are in agreement with morphological analyses of botiines, suggesting that Botia is the ancestral genus, while Leptobotia and Parabotia were resolved as more derived sister groups.     

Two-stage carbon distribution and cofactor generation for improving l-threonine production of Escherichia coli

L -Threonine, a kind of essential amino acid, has numerous applications in food, pharmaceutical, and aquaculture industries. Fermentative l -threonine production from glucose has been achieved in Escherichia coli. However, there are still several limiting factors hindering further improvement of l -threonine productivity, such as the conflict between cell growth and production, byproduct accumulation, and insufficient availability of cofactors (adenosine triphosphate, NADH, and NADPH). Here, a metabolic modification strategy of two-stage carbon distribution and cofactor generation was proposed to address the above challenges in E. coli THRD, an l -threonine producing strain. The glycolytic fluxes towards tricarboxylic acid cycle were increased in growth stage through heterologous expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and citrate synthase, leading to improved glucose utilization and growth performance. In the production stage, the carbon flux was redirected into l -threonine synthetic pathway via a synthetic genetic circuit. Meanwhile, to sustain the transaminase reaction for l -threonine production, we developed an l -glutamate and NADPH generation system through overexpression of glutamate dehydrogenase, formate dehydrogenase, and pyridine nucleotide transhydrogenase. This strategy not only exhibited 2.02- and 1.21-fold increase in l -threonine production in shake flask and bioreactor fermentation, respectively, but had potential to be applied in the production of many other desired oxaloacetate derivatives, especially those involving cofactor reactions.     

A novel adaptation of the integrin PSI domain revealed from its crystal structure

Integrin beta-subunits contain an N-terminal PSI (for plexin-semaphorin-integrin) domain that contributes to integrin activation and harbors the PI(A) alloantigen associated with immune thrombocytopenias and susceptibility to sudden cardiac death. Here we report the crystal structure of PSI in the context of the crystallized alphaVbeta3 ectodomain. The integrin PSI forms a two-stranded antiparallel beta-sheet flanked by two short helices; its long interstrand loop houses Pl(A) and may face the EGF2 domain. The integrin PSI contains four cysteine pairs connected in a 1-4, 2-8, 3-6, 5-7 pattern. An unexpected feature of the structure is that the final, eighth cysteine is located C-terminal to the Ig-like hybrid domain and is thus separated by the hybrid domain from the other seven cysteines of PSI. This architecture may be relevant to the evolution of integrins and should help refine the current models of integrin activation.