Fine mapping and candidate gene analysis of <Emphasis Type="Italic">hwh1</Emphasis> and <Emphasis Type="Italic">hwh2</Emphasis>, a set of complementary genes controlling hybrid breakdown in rice

Hybrid breakdown (HB), a phenomenon of reduced viability or fertility accompanied with retarded growth in hybrid progenies, often arises in the offspring of intersubspecific hybrids between indica and japonica in rice. We detected HB plants in F₈ recombinant inbred lines derived from the cross between an indica variety, Milyang 23, and a japonica variety, Tong 88-7. HB plants showed retarded growth, with fewer tillers and spikelets. Genetic analysis revealed that HB was controlled by the complementary action of two recessive genes, hwh1 and hwh2, originating from each of both parents, which were fine-mapped on the short arm of chromosome 2 and on the near centromere region of the long arm of chromosome 11, respectively. A comparison of the sequences of candidate genes among both parents and HB plants revealed that hwh1 encoded a putative glucose-methanol-choline oxidoreductase with one amino acid change compared to Hwh1 and that hwh2 probably encoded a putative hexose transporter with a six amino acid insertion compared to Hwh2. Investigation of the distribution of these alleles among 54 japonica and indica cultivars using candidate gene-based markers suggested that the two loci might be involved in developing reproductive barriers between two subspecies.     

Analysis of proteinase-activated receptor 2 and TLR4 signal transduction: a novel paradigm for receptor cooperativity

Proteinase-activated receptor 2 (PAR(2)), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR(2), often measured by Ca(2+) release. PAR(2) contributes to inflammation through activation of NF-kappaB-regulated genes; however, the mechanism by which this occurs is unknown. Overexpression of human PAR(2) in HEK293T cells resulted in concentration-dependent, PAR(2) AP-inducible NF-kappaB reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple G(i) proteins or sequester intracellular Ca(2+). Because previous studies described synergistic PAR(2)- and TLR4-mediated cytokine production, we hypothesized that PAR(2) and TLR4 might interact at the level of signaling. In the absence of TLR4, PAR(2)-induced NF-kappaB activity was inhibited by dominant negative (DN)-TRIF or DN-TRAM constructs, but not by DN-MyD88, findings confirmed using cell-permeable, adapter-specific BB loop blocking peptides. Co-expression of TLR4/MD-2/CD14 with PAR(2) in HEK293T cells led to a synergistic increase in AP-induced NF-kappaB signaling that was MyD88-dependent and required a functional TLR4, despite the fact that AP exhibited no TLR4 agonist activity. Co-immunoprecipitation of PAR(2) and TLR4 revealed a physical association that was AP-dependent. The response to AP or lipopolysaccharide was significantly diminished in TLR4(-/-) and PAR (-/-)(2) macrophages, respectively, and SW620 colonic epithelial cells exhibited synergistic responses to co-stimulation with AP and lipopolysaccharide. Our data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.     

Requirement of multiple protein domains and residues for gating K(ATP) channels by intracellular pH.

ATP-sensitive K(+) channels (K(ATP)) are regulated by pH in addition to ATP, ADP, and phospholipids. In the study we found evidence for the molecular basis of gating the cloned K(ATP) by intracellular protons. Systematic constructions of chimerical Kir6.2-Kir1.1 channels indicated that full pH sensitivity required the N terminus, C terminus, and M2 region. Three amino acid residues were identified in these protein domains, which are Thr-71 in the N terminus, Cys-166 in the M2 region, and His-175 in the C terminus. Mutation of any of them to their counterpart residues in Kir1.1 was sufficient to completely eliminate the pH sensitivity. Creation of these residues rendered the mutant channels clear pH-dependent activation. Thus, critical players in gating K(ATP) by protons are demonstrated. The pH sensitivity enables the K(ATP) to regulate cell excitability in a number of physiological and pathophysiological conditions when pH is low but ATP concentration is normal.     

Tetramethylpyrazine Protects Against Oxygen-Glucose Deprivation-Induced Brain Microvascular Endothelial Cells Injury via Rho/Rho-kinase Signaling Pathway

Tetramethylpyrazine (TMP, also known as Ligustrazine), which is isolated from Chinese Herb Medicine Ligustium wollichii Franchat (Chuan Xiong), has been widely used in China for the treatment of ischemic stroke by Chinese herbalists. Brain microvascular endothelial cells (BMECs) are the integral parts of the blood–brain barrier (BBB), protecting BMECs against oxygen-glucose deprivation (OGD) which is important for the treatment of ischemic stroke. Here, we investigated the protective mechanisms of TMP, focusing on OGD-injured BMECs and the Rho/Rho-kinase (Rho-associated kinases, ROCK) signaling pathway. The model of OGD-injured BMECs was established in this study. BMECs were identified by von Willebrand factor III staining and exposed to fasudil, or TMP at different concentrations (14.3, 28.6, 57.3 µM) for 2 h before 24 h of OGD injury. The effect of each treatment was examined by cell viability assays, measurement of intracellular reactive oxygen species (ROS), and transendothelial electric resistance and western blot analysis (caspase-3, endothelial nitric oxide synthase (eNOS), RhoA, Rac1). Our results show that TMP significantly attenuated apoptosis and the permeability of BMECs induced by OGD. In addition, TMP could notably down-regulate the characteristic proteins in Rho/ROCK signaling pathway such as RhoA and Rac1, which triggered abnormal changes of eNOS and ROS, respectively. Altogether, our results show that TMP has a strong protective effect against OGD-induced BMECs injury and suggest that the mechanism might be related to the inhibition of the Rho/ROCK signaling pathway.     

Trehalose Inhibits Protein Aggregation Caused by Transient Ischemic Insults Through Preservation of Proteasome Activity,Not via Induction of Autophagy

Protein aggregation has been proved to be a pathological basis accounting for neuronal death caused by either transient global ischemia or oxygen glucose deprivation (OGD), and inhibition of protein aggregation is emerging as a potential strategy of preventing brain damage. Trehalose was found to inhibit protein aggregation caused by neurodegenerative diseases via induction of autophagy, whereas its effect is still elusive on ischemia-induced protein aggregation. In this study, we investigated this issue by using rat model of transient global ischemia and SH-SY5Y model of OGD. We found that pretreatment with trehalose inhibited transient global ischemia-induced neuronal death in the hippocampus CA1 neurons and OGD-induced death in SH-SY5Y cells, which was associated with inhibition of the formation of ubiquitin-labeled protein aggregates and preservation of proteasome activity. In vitro study showed that the protection of trehalose against OGD-induced cell death and protein aggregation in SH-SY5Y cells was reversed when proteasome activity was inhibited by MG-132. Further studies revealed that trehalose prevented OGD-induced reduction of proteasome activity via suppression of both oxidative stress and endoplasmic reticulum stress. Particularly, our results showed that trehalose inhibited OGD-induced autophagy. Therefore, we demonstrated that proteasome dysfunction contributed to protein aggregation caused by ischemic insults and trehalose prevented protein aggregation via preservation of proteasome activity, not via induction of autophagy.     

The rapid degradation of bisphenol A induced by the response of indigenous bacterial communities in sediment

In the present study, sediment was spiked with bisphenol A (BPA) solution to explore the interaction between indigenous bacterial communities and BPA biodegradation in sediment. Results showed that BPA could be adsorbed to the sediment and then biodegraded rapidly. Biodegradation efficiency of BPA in treatments with 10 and 50 mg/L BPA reached 64.3 and 61.8% on the first day, respectively. Quantitative polymerase chain reaction and denaturing gradient gel electrophoresis analysis indicated that BPA affected the densities, species, and diversities of bacteria significantly. The response of bacterial community to BPA favored BPA biodegradation by promoting the growth of BPA-reducing bacteria and inhibiting other competitors. According to the results of sequencing, Pseudomonas and Sphingomonas played vital roles in the degradation of BPA. They presented over 73% of the original bacterial community, and both of them were promoted by BPA comparing with controls. Laccase and polyphenol oxidase contributed to the degradation of BPA and metabolic intermediates, respectively. This paper illustrates the rapid biodegradation of BPA induced by the response of indigenous bacterial communities to the BPA stress, which will improve the understandings of BPA degradation in sediment.

     

Synthesis and anti-tumor evaluation of panaxadiol halogen-derivatives

In the current work, 13 novel panaxadiol (PD) derivatives were synthesized by reacting with chloroacetyl chloride and bromoacetyl bromide. Their in vitro antitumor activities were evaluated on three human tumor cell lines (HCT-116, BGC-823, SW-480) and three normal cells (human gastric epithelial cell line-GES-1, hair follicle dermal papilla cell line-HHDPC and rat myocardial cell line-H9C2) by MTT assay. Compared with PD, the results demonstrated that compound 1e, 2d, 2e showed significant anti-tumor activity against three tumor cell lines, the IC50 value of compound 2d against HCT-116 was the lowest (3.836 μM). The anti-tumor activity of open-ring compounds are significantly better than the compounds of C-25 cyclization. Compound 1f, 2f, 2g showed the strong anti-tumor activity. The IC50 value of compound 2g against BGC-823 and SW-480 were the lowest (0.6 μM and 0.1 μM, respectively). Combined with cytotoxicity test, the IC50 value of compound 1e, 2d, 2e are greater than 100. the open-ring compounds (1f, 2f, 2g) showed a strong toxicity. The toxicity of 1f is lower than 2f and 2g. These compounds may be useful for the development of novel antiproliferative agents.     

Optimized culture medium for the production of flavonoids from <Emphasis Type="Italic">Orostachys cartilaginea</Emphasis> V.N. Boriss. callus cultures

The optimal culture medium for the production of flavonoid compounds from Orostachys cartilaginea V. N. Boriss. calluses was studied. In callus cultures of O. cartilaginea, the flavonoid monomer content, in decreasing order was kaempferol-3-O-rutinoside (Kp-3-rut), quercetin 3-O-glucoside (Qc-3-glc), epicatechin gallate (Ecg), kaempferide (Ke), and quercetin (Qc). The results of the uniform design experiment indicated that the production of Qc, Ke, Qc-3-glc, Kp-3-rut, and total flavonoids were satisfactory in callus grown on full salt strength (1×) of Murashige and Skoog (MS) medium supplemented with 3.5 mg L^?1 6-benzylaminopurine (BA) and 0.1 mg L^?1 1-naphthalene acetic acid (NAA). By contrast, only Ecg was found in callus grown on 0.75× MS medium supplemented with 1.5 mg L^?1 BA and 0.3 mg L^?1 NAA. A phosphate concentration of 1.25 mM in the MS medium favored the production of Qc and Ke, whereas 0.75 mM phosphate was optimal for the production of Ecg, Qc-3-glc, Kp-3-rup, and total flavonoids. The NH₄ ⁺/NO₃ ^? ratios of 30/30 mM in the MS medium promoted Ke, Ecg, Qc-3-glc, Kp-3-rup, and total flavonoid production. However, a NH₄ ⁺/NO₃ ^? ratio of 20/40 mM enhanced Qc production. The effect of sucrose concentrations on the accumulation of different flavonoid monomers was comparatively more regular. The flavonoid content increased as the sucrose concentration increased from 20 to 40 g L^?1, peaked at 40 g L^?1, and decreased at concentrations greater than 40 g L^?1. Therefore, 40 g L^?1 sucrose was optimal for the production of the five flavonoid monomers and total flavonoids. The present findings demonstrate the possibility of producing flavonoid compounds from O. cartilaginea callus.     

三种萤火虫幼虫臀足的形态与功能

本文利用显微拍照和电镜扫描技术,以黄缘窗萤(Pyrocoelia analis)、穹宇萤(Pygoluciola qingyu)和雷氏黄萤(Aquatica leii)为例,对四川峨眉山较为常见的三种萤火虫幼虫臀足的形态结构及功能进行了研究。研究表明,三种萤火虫幼虫臀足的相似特征为:腹内侧均有趾钩,趾钩的排布规律基本相同;均为左右对称的两部分,背外侧的臀足较长,腹内侧的臀足较短;基本都为白色透明。不同特征为:黄缘窗萤幼虫的臀足数量最多,穹宇萤次之,雷氏黄萤最少;黄缘窗萤幼虫的臀足存在一级或两级的分叉现象,而另外两种则无此现象;臀足基部的斑纹和刚毛的情况各有不同,差异较大。三种萤火虫幼虫臀足都有着相似的功能:吸附功能,将身体吸附在物体上;作为清理工具,清理体表黏着的泥土等脏物;辅助爬行。此外,穹宇萤幼虫还可利用臀足筑巢化蛹。     

A novel B-2 suppressor cell regulating susceptibility/resistance of mice to Toxoplasma gondii infection

Irradiation treatment enhanced resistance of C57BL/6, but not BALB/c against Toxoplasma gondii infection. Six Gy-irradiated (IR) C57BL/6 recipients of B-2 cells from T. gondii-infected C57BL/6 died after infection. B-2 suppressor cells from infected C57BL/6 enhanced production of IL-4 and IL-10 in peritoneal exudate cells (PECs), and down-regulated NO release in peritoneal macrophages after infection. On the other hand, B-2 suppressor cells were not detected in a strain, BALB/c, resistant against infection. These data indicated that irradiation-sensitive B-2 cells regulated susceptibility/resistance in mice against T. gondii infection.