Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.     

Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4

Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane.Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells was dependent on added Wnt5A protein and was accomplished by the multifunctional adaptor protein beta-arrestin 2 (betaarr2), which was recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of beta-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling.     

Immunocyte Ca2+ influx system mediated by LTRPC2

We characterized an activation mechanism of the human LTRPC2 protein, a member of the transient receptor potential family of ion channels, and demonstrated that LTRPC2 mediates Ca2+ influx into immunocytes. Intracellular pyrimidine nucleotides, adenosine 5'-diphosphoribose (ADPR), and nicotinamide adenine dinucleotide (NAD), directly activated LTRPC2, which functioned as a Ca2+-permeable nonselective cation channel and enabled Ca2+ influx into cells. This activation was suppressed by intracellular adenosine triphosphate. These results reveal that ADPR and NAD act as intracellular messengers and may have an important role in Ca2+ influx by activating LTRPC2 in immunocytes.     

Integrins regulate Rac targeting by internalization of membrane domains

Translocation of the small GTP-binding protein Rac1 to the cell plasma membrane is essential for activating downstream effectors and requires integrin-mediated adhesion of cells to extracellular matrix. We report that active Rac1 binds preferentially to low-density, cholesterol-rich membranes, and specificity is determined at least in part by membrane lipids. Cell detachment triggered internalization of plasma membrane cholesterol and lipid raft markers. Preventing internalization maintained Rac1 membrane targeting and effector activation in nonadherent cells. Regulation of lipid rafts by integrin signals may regulate the location of membrane domains such as lipid rafts and thereby control domain-specific signaling events in anchorage-dependent cells.     

Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity

Rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1), extends the life spans of yeast, flies, and mice. Calorie restriction, which increases life span and insulin sensitivity, is proposed to function by inhibition of mTORC1, yet paradoxically, chronic administration of rapamycin substantially impairs glucose tolerance and insulin action. We demonstrate that rapamycin disrupted a second mTOR complex, mTORC2, in vivo and that mTORC2 was required for the insulin-mediated suppression of hepatic gluconeogenesis. Further, decreased mTORC1 signaling was sufficient to extend life span independently from changes in glucose homeostasis, as female mice heterozygous for both mTOR and mLST8 exhibited decreased mTORC1 activity and extended life span but had normal glucose tolerance and insulin sensitivity. Thus, mTORC2 disruption is an important mediator of the effects of rapamycin in vivo.     

Thalamic control of visceral nociception mediated by T-type Ca2+ channels

Sensations from viscera, like fullness, easily become painful if the stimulus persists. Mice lacking alpha1G T-type Ca2+ channels show hyperalgesia to visceral pain. Thalamic infusion of a T-type blocker induced similar hyperalgesia in wild-type mice. In response to visceral pain, the ventroposterolateral thalamic neurons evokeda surge of single spikes, which then slowly decayed as T type-dependent burst spikes gradually increased. In alpha1G-deficient neurons, the single-spike response persisted without burst spikes. These results indicate that T-type Ca2+ channels underlie an antinociceptive mechanism operating in the thalamus andsupport the idea that burst firing plays a critical role in sensory gating in the thalamus.     

BIBAC2载体转化水稻的研究

双元细菌人工染色体(BIBAC)载体具有克隆大片段DNA构建文库和农杆菌介导转化植物的双重功能.利用BIBAC载体,在双子叶植物烟草和番茄中已实现了大片段DNA的转移,但在单子叶植物中的应用还未见报道.本研究利用不同水稻基因型、不同农杆菌菌株,对水稻进行了BIBAC2载体的转化研究.水稻成熟胚诱导愈伤组织经过1~2次继代培养,在含乙酰丁香酮的N6培养基上预培养3 d,在含BIBAC2载体的农杆菌中侵染10 min,26℃下共培养3 d.感染后的愈伤组织在含25~50 mg/L潮霉素培养基上经过4~8周的筛选,共得到了66株抗性再生植株.通过对抗性植株中GUS基因的组织化学染色检测,NPTII基因的PCR检测和ELISA检测,HYG基因的PCR检测,确认2株阳性植株来自EHA105菌系侵染的中花15号愈伤组织,1株阳性植株来自EHA105菌系侵染的中花8号愈伤组织,转化率分别为2.9%和0.9%.结果表明,BIBAC2载体已被成功导入了水稻基因组中,转化效果EHA105菌株优于C58C1菌株,中花15号、中花8号优于中花10号和中花11号.结果还表明,利用三亲交配法可以将大于23.5 kb的载体转化到农杆菌中.初步建立了BIBAC2载体转化水稻的技术体系,为利用BIBAC系统向水稻转入大片段DNA和多个外源功能基因奠定了基础.     

Long-term potentiation of neuron-glia synapses mediated by Ca2+-permeable AMPA receptors

Interactions between neurons and glial cells in the brain may serve important functions in the development, maintenance, and plasticity of neural circuits. Fast neuron-glia synaptic transmission has been found between hippocampal neurons and NG2 cells, a distinct population of macroglia-like cells widely distributed in the brain. We report that these neuron-glia synapses undergo activity-dependent modifications analogous to long-term potentiation (LTP) at excitatory synapses, a hallmark of neuronal plasticity. However, unlike the induction of LTP at many neuron-neuron synapses, both induction and expression of LTP at neuron-NG2 synapses involve Ca2+-permeable AMPA receptors on NG2 cells.     

巨噬细胞介导PCV2诱导体外培养仔猪淋巴细胞凋亡的研究

选用5头猪圆环病毒2型(PCV2)及猪呼吸和繁殖综合征病毒(PRRSV)抗体阴性普通断奶仔猪,无菌分离脾脏淋巴细胞和巨噬细胞(macrophage,Mφ),分别以PCV2和PCV2+Mφ与淋巴细胞相互作用,同时设Mφ和淋巴细胞对照组.培养0、6、12、24 h后分别收取各组淋巴细胞,用流式细胞术对细胞凋亡率进行测定,半定量RT-PCR法测定淋巴细胞Fas和FasL mRNA的转录情况.结果显示:PCV2+Mφ组的淋巴细胞凋亡率在24 h时显著高于其他3组(P<0.05).PCV2+Mφ组的淋巴细胞6 h时Fas mRNA转录量显著高于空白对照组(P<0.05),24 h时显著高于其他3组(P<0.05).FasL mRNA的转录变化与Fas mRNA基本一致.相关性分析表明,淋巴细胞凋亡率和Fas(FasL)mRNA的转录呈极显著相关(Fas:P=0.000,r=0.606;FasL:P=0.000,r=0.579).表明:巨噬细胞能介导PCV2诱导体外培养淋巴细胞的凋亡,这种细胞凋亡的发生与Fas/FasL系统紧密相关.     

地熊蜂非视觉相关beta-arrestin基因的克隆与表达谱分析

[目的]克隆鉴定地熊蜂非视觉相关beta-arrestin基因并分析其在蜂王不同发育时期及特定组织中的表达差异,以期为该基因的功能研究奠定基础。[方法]利用PCR扩增和基因克隆技术获得地熊蜂非视觉相关beta arrestin基因的cDNA全长序列;利用生物信息学技术对该基因编码的氨基酸序列进行比对和进化树分析;运用Real-time PCR技术分析该基因在地熊蜂蜂王出房前1天、出房第1天、交配后5天、越冬3个月、越冬5个月、产卵第1天和饲养3个月后的脑、卵巢、中肠和脂肪体表达情况。[结果]beta-arrestin基因CDS序列长1 248bp,编码的蛋白大小为415aa。该蛋白氨基酸序列中第27～182个氨基酸残基为Arrestin-N superfamily的保守结构域;第201～354个氨基酸残基为Arrestin-Csuperfamily的保守结构域。相似性分析表明,地熊蜂beta-arrestin基因氨基酸序列与NCBI中收录的地熊蜂转录组数据的氨基酸序列一致性高达97%。Real-time PCR结果表明,beta-arrestin基因在地熊蜂所有检测的发育阶段及特定组织均有所表达,但表达量差异很大(P0.05)。其中在头部和中肠表达量较高的时期是越冬3个月蜂王,在卵巢和脂肪体表达量较高的时期是出房第1天蜂王。[结论]本研究克隆了地熊蜂beta-arrestin基因的CDS序列,确定了beta-arrestin基因CDS序列编码的氨基酸序列,分析了该基因的序列特征和表达谱,推测其与蜂王越冬期间生理代谢过程和蜂王性成熟阶段激素分泌活动有关。     

Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization

Hepcidin is a peptide hormone secreted by the liver in response to iron loading and inflammation. Decreased hepcidin leads to tissue iron overload, whereas hepcidin overproduction leads to hypoferremia and the anemia of inflammation. Ferroportin is an iron exporter present on the surface of absorptive enterocytes, macrophages, hepatocytes, and placental cells. Here we report that hepcidin bound to ferroportin in tissue culture cells. After binding, ferroportin was internalized and degraded, leading to decreased export of cellular iron. The posttranslational regulation of ferroportin by hepcidin may thus complete a homeostatic loop: Iron regulates the secretion of hepcidin, which in turn controls the concentration of ferroportin on the cell surface.     

根癌农杆菌介导的甘蔗遗传转化

综述开展甘蔗基因工程育种工作20多年来,从最初的利用农杆菌介导法研究抗除草剂和抗虫转基因成功,到目前高转化效率的转基因方法应用过程中取得的成果,分析影响农杆菌转化甘蔗的关键因素,并对农杆菌介导的甘蔗遗传转化研究前景作了展望.     

农杆菌介导的遗传转化在花生上的应用及前景

农杆菌介导的遗传转化已广泛应用于各种植物。本文综述了农杆菌介导的遗传转化在花生上的应用及其前景。     

Genomic sequencing and methylation analysis by ligation mediated PCR

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited because of the complexity of the mammalian genome. A newly developed genomic sequencing procedure in which a ligation mediated polymerase chain reaction (PCR) is used generates high quality, reproducible sequence ladders starting with only 1 microgram of uncloned mammalian DNA per reaction. Different sequence ladders can be created simultaneously by inclusion of multiple primers and visualized separately by rehybridization. Relatively little radioactivity is needed for hybridization and exposure times are short. Methylation patterns in genomic DNA are readily detectable; for example, 17 CpG dinucleotides in the 5' region of human X-linked PGK-1 (phosphoglycerate kinase 1) were found to be methylated on an inactive human X chromosome, but unmethylated on an active X chromosome.     

Ongoing modification of Mediterranean Pleistocene sapropels mediated by prokaryotes

Late Pleistocene organic-rich sediments (sapropels) from the eastern Mediterranean Sea harbor unknown, metabolically active chemoorganotrophic prokaryotes. As compared to the carbon-lean intermediate layers, sapropels exhibit elevated cell numbers, increased activities of hydrolytic exoenzymes, and increased anaerobic glucose degradation rates, suggesting that microbial carbon substrates originate from sapropel layers up to 217,000 years old. 16S ribosomal RNA gene analyses revealed that as-yet-uncultured green nonsulfur bacteria constitute up to 70% of the total microbial biomass. Crenarchaeota constitute a smaller fraction (on average, 16%). A slow but significant turnover of glucose could be detected. Apparently, sapropels are still altered by the metabolic activity of green nonsulfur bacteria and crenarchaeota.     

Selective trafficking of non-cell-autonomous proteins mediated by NtNCAPP1

In plants, cell-to-cell communication is mediated by plasmodesmata and involves the trafficking of non-cell-autonomous proteins (NCAPs). A component in this pathway, Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (NtNCAPP1), was affinity purified and cloned. Protein overlay assays and in vivo studies showed that NtNCAPP1 is located on the endoplasmic reticulum at the cell periphery and displays specificity in its interaction with NCAPs. Deletion of the NtNCAPP1 amino-terminal transmembrane domain produced a dominant-negative mutant that blocked the trafficking of specific NCAPs. Transgenic tobacco plants expressing this mutant form of NtNCAPP1 and plants in which the NtNCAPP1 gene was silenced were compromised in their ability to regulate leaf and floral development. These results support a model in which NCAP delivery to plasmodesmata is both selective and regulated.     

发根农杆菌介导的杨梅遗传转化研究初报

为了建立发根农杆菌Agrobacterium rhizogenes介导的杨梅Myrica rubra遗传转化体系,利用携带拟南芥Arabidopsis thaliana LEAFYcDNA片段的发根农杆菌R15834感染东魁杨梅M. rubra‘Dongkui’和荸荠杨梅M. rubra‘Boji’的子叶、叶片和茎段。结果表明,只从东魁杨梅子叶上诱导出毛状根,而且产生毛状根的东魁杨梅子叶数在10%以下。杨梅子叶产生毛状根受多种因素影响,新种子比旧种子容易产生毛状根,在1/2 MS(Murashige and Skoog)培养基培养较在MS培养基上培养好。东魁杨梅子叶上的毛状根经聚合酶链反应(polymerase chain reaction,PCR)检测和Southern杂交检测,证实已将拟南芥的LEAFYcDNA片段成功导入并整合到东魁杨梅子叶中。图2参10