Sphingosine kinase type 1 induces G12/13-mediated stress fiber formation,yet promotes growth and survival independent of G protein-coupled receptors

Sphingosine 1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors (GPCRs) that regulate a wide variety of important cellular functions, including growth, survival, cytoskeletal rearrangements, and cell motility. However, whether it also has an intracellular function is still a matter of great debate. Overexpression of sphingosine kinase type 1, which generated S1P, induced extensive stress fibers and impaired formation of the Src-focal adhesion kinase signaling complex, with consequent aberrant focal adhesion turnover, leading to inhibition of cell locomotion. We have dissected biological responses dependent on intracellular S1P from those that are receptor-mediated by specifically blocking signaling of Galphaq, Galphai, Galpha12/13, and Gbetagamma subunits, the G proteins that S1P receptors (S1PRs) couple to and signal through. We found that intracellular S1P signaled "inside out" through its cell-surface receptors linked to G12/13-mediated stress fiber formation, important for cell motility. Remarkably, cell growth stimulation and suppression of apoptosis by endogenous S1P were independent of GPCRs and inside-out signaling. Using fibroblasts from embryonic mice devoid of functional S1PRs, we also demonstrated that, in contrast to exogenous S1P, intracellular S1P formed by overexpression of sphingosine kinase type 1 promoted growth and survival independent of its GPCRs. Hence, exogenous and intracellularly generated S1Ps affect cell growth and survival by divergent pathways. Our results demonstrate a receptor-independent intracellular function of S1P, reminiscent of its action in yeast cells that lack S1PRs.     

Mesenchymal Stem Cell Transplantation Enhancement in Myocardial Infarction Rat Model under Ultrasound Combined with Nitric Oxide Microbubbles

Objective

This study evaluated the effects of ultrasound combined with the homemade nitric oxide (NO) micro-bubble destruction on the in vitro proliferation, apoptosis, and migration of mesenchymal stem cells (MSCs). Furthermore, we studied whether or not irradiation of the NO micro-bubble combined with bone-marrow derived MSC infusion had a better effect on treating myocardial infarction. The possible mechanism of MSC delivery into the infarcted myocardium was also investigated.

Methods

The murine bone marrow-derived MSCs were isolated, cultured, irradiated, and combined with different concentrations of NO microbubbles. MTT proliferation assay, annexin V-FITC apoptosis detection, migration assay, and RT-PCR were performed 24 h after the irradiation. The NO micro-bubbles was a intravenously injected, followed by the infusion of MSCs, which were labeled by CM-Dil. Myocardium was harvested 48 h later and the distribution of MSCs was observed by laser scanning confocal microscope after frozen sectioning. Echocardiography, histological examination, RT-PCR, and western blotting were performed four weeks after the cell transplantation.

Results

Ultrasound combined with 1:70 NO micro-bubbles had no significant impact on the proliferation or apoptosis of MSCs. Transwell chamber findings demonstrated that MSCs migrated more efficiently in group that underwent ultrasound combined with 1:70 NO micro-bubbles. The Real-time PCR results indicated that the expression of CXCR4 was much higher in the group undergoing ultrasound combined with 1:70 NO micro-bubbles. The normalized fluorescence intensity greatly increased in the group of US+NO micro-bubbles and the cardiac function was also markedly improved. Immunohistochemical staining showed that the capillary density was much greater in the group of US+NO micro-bubbles as compared to that of the other groups. RT-PCR and western blotting also revealed a higher SDF-1 and VEGF expression in the group of US+NO micro-bubbles.

Conclusions

NO micro-bubbles could be used in the cell transplantation, which efficiently promoted the MSC homing into the infarcted myocardium.     

Molecular characterization of sawtooth oak (Quercus acutissima) germplasm based on randomly amplified polymorphic DNA

Sawtooth oak (Quercus acutissima) is a predominant tree species in the deciduous broad-leaved forest in China. It distributes in a large landscape area and can disperse in various ecology types. Molecular study on sawtooth oak can provide valuable information about the genetic diversity level and genetic relatedness on this important tree species. Insight into the genetic structure also provides resources of a species with its current feature and future evolutionary potential. The genetic structure of sawtooth oak was investigated by randomly amplified polymorphic DNA (RAPD). Twelve RAPD markers were used to assess genetic diversity of 408 individuals from 17 provenances enveloping most of the current distribution area of sawtooth oak. A total of 66 amplification products were detected, of which 49 bands (74.24 %) were polymorphic. Nei’s gene diversity, 0.2409, indicated a relatively high level of genetic variation in sawtooth oak germplasm. Analysis of molecular variance showed that most of the genetic diversity (87 %) was allocated within provenances. A combination of UPGMA dendrogram and STRUCTURE analysis was employed to estimate the genetic relationships of sawtooth oak germplasm; interestingly, the two methods presented similar grouping pattern with few discrepancies. Results revealed that 16 out of 17 provenances were clustered into one group, while the other 1 (LQ provenance) constituted a separate cluster. The data presented in this study suggested that the RAPD method was a valuable tool for estimation of genetic diversity and genetic relatedness of sawtooth oak germplasm. The present study also gave useful implications for germplasm conservation and new cultivar development for this promising energy tree species.     

北沙参中佛手柑内酯的分离鉴定及体外抗肿瘤活性的初步测定

北沙参为传统的中药材,来源于伞形科(Umbelliferae)植物珊瑚菜(Glehnia littoralis Fr. Schmidt ex Miq. )的干燥根,具有养阴清肺、益胃生津的功效[1],其道地产地为山东莱阳.     

Species composition and diversity of soil animals in the oil shale dump in Maoming, Guangdong Province, China

Pollution from the oil shale dump in Maoming, Guangdong Province, China, was a major social problem due to the great amount of environmental damage it caused. Therefore, a great deal of attention needed to be paid for the ecological restoration and reconstruction. The objective of this study was to investigate the species composition of soil fauna and its diversity in oil shale dumps after the application of different ecological restoration schemes in order to understand the impact they had on ecological restoration. Three plots were selected on an oil shale dump near the city of Maoming. The “north plot” was a newly-planted young forest mixed with various tree species, while the “south plot” was a 20-year-old Acacia auriculaeformis forest, and the “control plot” was a 20-year-old naturally-recovering grassland. Soil animals, mainly including macro-meso groups, were collected by hand-sorting and Tullgren funnel extraction. They were then identified to family or genus level with only a small portion to order (e.g. Chllopoda) or species (e.g. Isopoda) level. The specimens obtained in the present study was 11164 individuals, belonging to 27 orders and 110 families or genera. The Shannon index (H′), DG_s (based on species) and DG_g (based on groups) were used to analyze the diversity of soil animals between different plots. The major results were as follows: A total of 33 families or genera belonging to nine orders were found in the “north plot”. The main group was Caritermes, accounting for 63.4% of the total, followed by Tetramorium with 21.3%. Hymenoptera, mainly Formicidae, had more genera than others, accounting for 80% of the total genera in this group. The diversity of soil animals in this plot was very low because the H′ index was only 1.2, while the DG_s index was 4.0 and the DG_g index was 1.3. A total of 61 families or genera belonging to 23 orders were found in the “south plot”. Malmcoangelia and Tetramorium were the main groups, which accounted for 60.3% and 10.2%, respectively. Two genera of Annelda and two genera of Isopoda only accounted for 2.6% and 1.9%, respectively, but they were considered to be major groups due to their large body sizes and the distinct characteristics of their habitat. Acarina had a greater number of individuals and families or genera with its individual number accounting for 67.5% of the total, and the number of families or genera of this group account for 70% in this plot. The diversity indexes (H′, DG_s and DG_g) in this plot were significantly higher than those in the “north plot”, and were 1.65, 16.7 and 7.75, respectively. In the “control plot”, there were 67 families or genera of soil animals belonging to 23 orders. The main groups were Tetramorium (20%), Lasius (17.1%), Bothriomymex of Formicidae (13.8%), and Malmcoangelia of oribatid mites (14.5%). Formicidae of Hymenoptera was the group with the maximum number of individuals, accounting for 51.0%, while Diplopoda had the most families or genera. The H′ and DG_s indexes, being 2.54 and 17.7, were higher than those in the “south plot”, while the DG_g index of 7.20 was lower than that in the “south plot”. The results showed that the species composition and diversity indexes were higher in the “south plot” than in the “north plot” and the “control plot”, which demonstrated that using Acacia auriculaeformis forest to restore the oil shale dump was an effective approach in terms of soil biodiversity.     

杜氏盐藻rbcS启动子的克隆和功能分析

为提高转基因盐藻的表达效率,利用基因组步行方法和巢式PCR,从盐藻中克隆了1,5-二磷酸核酮糖羧化酶/加氧酶（Rubisco）的小亚基基因rbcS 的5'上游调控序列,并对其进行序列分析和转基因功能分析。采用Dra I、EcoR V、Pvu II和Stu I四种平端限制内切酶分别酶切盐藻基因组DNA,并与接头连接,构建基因组步行文库GWL 1、GWL 2、GWL 3和GWL 4;设计特异引物从这四种文库中扩增rbcS基因的5'上游调控序列。在GWL 1、GWL 4中分别扩增出约1.2 kb的片段。对该序列的分析表明,它的3'端与已知盐藻rbcS cDNA 的5'端序列完全一致,说明是该基因的5'端上游区,并且包含多个与转录调控有关的保守序列（如TATA-box、CAAT-box）,富含GT的重复序列。此序列EcoR I下游的片段与除草剂抗性基因bar相融合,构建表达载体,电击法转化盐藻。通过对转化藻株的抗性筛选以及PCR和Southern blot检测,表明该区域能驱动外源基因bar在转基因盐藻中的表达,推断是盐藻rbcS基因的启动子调控区。     

Tracing the transition of methicillin resistance in sub-populations of Staphylococcus aureus, using SELDI-TOF Mass Spectrometry and Artificial Neural Network Analysis

Strains (n = 99) of Staphylococcus aureus isolated from a large number of clinical sources and tested for methicillin sensitivity were analysed by MALDI-TOF-MS using the Weak Cation Exchange (CM10) ProteinChip Array (designated SELDI-TOF-MS). The profile data generated was analysed using Artificial Neural Network (ANN) Analysis modelling techniques. Seven key ions identified by the ANNs that were predictive of MRSA and MSSA were validated by incorporation into a model. This model exhibited an area under the ROC curve value of 0.9147 indicating the potential application of this approach for rapidly characterising MRSA and MSSA isolates. Nearly all strains (n = 97) were correctly assigned to the correct group, with only two aberrant MSSA strains being misclassified. However, approximately 21% of the strains appeared to be in a process of transition as resistance to methicillin was being acquired.     

The Bryopsis hypnoides plastid genome: multimeric forms and complete nucleotide sequence

Background

Bryopsis hypnoides Lamouroux is a siphonous green alga, and its extruded protoplasm can aggregate spontaneously in seawater and develop into mature individuals. The chloroplast of B. hypnoides is the biggest organelle in the cell and shows strong autonomy. To better understand this organelle, we sequenced and analyzed the chloroplast genome of this green alga.

Principal Findings

A total of 111 functional genes, including 69 potential protein-coding genes, 5 ribosomal RNA genes, and 37 tRNA genes were identified. The genome size (153,429 bp), arrangement, and inverted-repeat (IR)-lacking structure of the B. hypnoides chloroplast DNA (cpDNA) closely resembles that of Chlorella vulgaris. Furthermore, our cytogenomic investigations using pulsed-field gel electrophoresis (PFGE) and southern blotting methods showed that the B. hypnoides cpDNA had multimeric forms, including monomer, dimer, trimer, tetramer, and even higher multimers, which is similar to the higher order organization observed previously for higher plant cpDNA. The relative amounts of the four multimeric cpDNA forms were estimated to be about 1, 1/2, 1/4, and 1/8 based on molecular hybridization analysis. Phylogenetic analyses based on a concatenated alignment of chloroplast protein sequences suggested that B. hypnoides is sister to all Chlorophyceae and this placement received moderate support.

Conclusion

All of the results suggest that the autonomy of the chloroplasts of B. hypnoides has little to do with the size and gene content of the cpDNA, and the IR-lacking structure of the chloroplasts indirectly demonstrated that the multimeric molecules might result from the random cleavage and fusion of replication intermediates instead of recombinational events.