Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis

The amino acid sequences of ten proteins with intracellular half-lives less than 2 hours contain one or more regions rich in proline (P), glutamic acid (E), serine (S), and threonine (T). These PEST regions are generally, but not always, flanked by clusters containing several positively charged amino acids. Similar inspection of 35 proteins with intracellular half-lives between 20 and 220 hours revealed that only three contain a PEST region. On the basis of this information, it was anticipated that caseins, which contain several PEST sequences, would be rapidly degraded within eukaryotic cells. This expectation was confirmed by red blood cell-mediated microinjection of 125I-labeled caseins into HeLa cells where they exhibited half-lives of less than 2 hours. The rapid degradation of injected alpha- and beta-casein as well as the inverse correlation of PEST regions with intracellular stability indicate that the presence of these regions can result in the rapid intracellular degradation of the proteins containing them.     

丝瓜络固定化微生物对土壤多环芳烃吸附-降解作用

以假单胞菌(Pseudomonas sp.SDR4,简称S4)、毛霉真菌(Mucormucedo sp.SDR1,简称S1)为研究对象,采用微生物固定化技术,研究了其对土壤多环芳烃的吸附和降解动力学,并探讨了固定化微生物对土壤多环芳烃的吸附机理及吸附降解关系。结果表明:试验60 d,改性丝瓜络(CK)、死体固定化S1(S1-D)、死体固定化S4(S4-D)、死体固定化S1与S4混合菌(S1+S4-D)对菲(Phe)的动态平衡吸附量分别为5.28、6.82、5.73、7.46μg,对芘(Pyr)的动态平衡吸附量分别为4.17、4.72、4.53、5.00μg,死体固定化微生物对Phe与Pyr的吸附过程均服从于准二级动力学;活体真菌S1、细菌S4、混合菌S1+S4对Phe的动态吸附量分别为2.32、2.01、2.76μg,对Pyr的动态吸附量分别为2.79、2.41、3.14μg,活体固定化微生物对土壤中Phe与Pyr的准一级动力学与准二级动力学拟合结果R2相差较小;S1、S4、S1+S4对Phe的降解率分别为54.34%、61.45%、64.23%,对Pyr的降解率分别为38.42%、35.02%、42.43%;经S1、S4、S1+S4处理后,Phe的降解半衰期分别为38.88、29.41、25.63 d,Pyr的降解半衰期分别为64.76、69.02、59.28 d。研究表明,化学作用是控制丝瓜络固定化微生物对多环芳烃吸附速率的主要因素;提高微生物的降解能力能增加对土壤中PAHs迁移的影响;混合菌中真菌与细菌存在协同作用,能提高Phe与Pyr的降解效率。     

金霉素及其异构体降解产物对斜生栅藻的毒性效应研究

为探索水体中四环素类抗生素异构体降解产物的毒性效应,选取金霉素及其异构体降解产物为目标化合物,斜生栅藻(Scenedesmus obliquus)为受试生物,结合藻类生理指标探讨金霉素及其异构体降解产物的毒性效应。研究结果表明,在斜生栅藻藻液中,金霉素主要的异构体降解产物为异金霉素及异差向金霉素。暴露72 h后,金霉素及其异构体降解产物处理组的藻细胞形态发生了明显的质壁分离,且细胞通透性均显著增大。但是,金霉素母体药物与不同异构体降解产物对斜生栅藻的毒性效应表现出了明显的差异。金霉素母体药物处理组的斜生栅藻细胞中可溶性蛋白质和叶绿素a浓度降低得更为显著,且氧化损伤更为严重。金霉素母体药物与其异构体降解产物虽然具有相似的化学结构,但取代基空间构象的不同会导致药物与藻细胞中的可溶性蛋白质的结合位点不同,因而对藻细胞产生不同的毒性效应。探究金霉素及其异构体降解产物对水生环境产生的毒性效应,有望为全面认识四环素类抗生素的生态环境风险提供新的依据。     

Specificity and stability in topology of protein networks

Molecular networks guide the biochemistry of a living cell on multiple levels: Its metabolic and signaling pathways are shaped by the network of interacting proteins, whose production, in turn, is controlled by the genetic regulatory network. To address topological properties of these two networks, we quantified correlations between connectivities of interacting nodes and compared them to a null model of a network, in which all links were randomly rewired. We found that for both interaction and regulatory networks, links between highly connected proteins are systematically suppressed, whereas those between a highly connected and low-connected pairs of proteins are favored. This effect decreases the likelihood of cross talk between different functional modules of the cell and increases the overall robustness of a network by localizing effects of deleterious perturbations.     

A peptide sequence confers retention and rapid degradation in the endoplasmic reticulum

A nonlysosomal pathway exists for the degradation of newly synthesized proteins retained within the endoplasmic reticulum (ER). This pathway is extremely selective: whereas some proteins are rapidly degraded, others survive for long periods in the ER. The question of whether this selectivity is due to the presence within the sensitive proteins of definable peptide sequences that are sufficient to target them for degradation has been addressed. Deletion of a carboxyl-terminal sequence, comprising the transmembrane domain and short cytoplasmic tail of the alpha chain of the T cell antigen receptor (TCR-alpha), prevented the rapid degradation of this polypeptide. Fusion of this carboxyl-terminal sequence to the extracellular domain of the Tac antigen, a protein that is normally transported to the cell surface where it survives long-term, resulted in the retention and rapid degradation of the chimeric protein in the ER. Additional mutagenesis revealed that the transmembrane domain of TCR-alpha alone was sufficient to cause degradation within the ER. This degradation was not a direct consequence of retention in the ER, as blocking transport of newly synthesized proteins out of the ER with brefeldin A did not lead to degradation of the normal Tac antigen. It is proposed that a 23-amino acid sequence, comprising the transmembrane domain of TCR-alpha, contains information that determines targeting for degradation within the ER system.     

Dopamine-accumulating retinal neurons revealed by in vitro fluorescence display a unique morphology

Dopamine is the principal catecholamine neurotransmitter in the vertebrate retina. The shape of retinal neurons that accumulate dopamine has been demonstrated in an in vitro preparation of cat retina. This was achieved by the discovery that the combined uptake of dopamine and the indoleaminergic transmitter analog 5,7-dihydroxytryptamine leads to an intense, catecholamine-like fluorescence in the cell bodies and processes of presumed dopaminergic amacrine cells in the living retina. This fluorescence served as an in vitro marker for these cells, and their detailed morphology was analyzed after intracellular injection of horseradish peroxidase under direct microscopic control. The horseradish peroxidase-filled cells show an unprecedented neuronal morphology: each cell gives rise to multiple, axon-like processes that arise from, and extend for millimeters beyond, the dendritic tree. The unique structure of this type of amacrine cell suggests a function for dopamine in long-range lateral interactions in the inner plexiform layer.     

微塑料在菲降解过程中对融合菌株F14的影响

以聚乙烯微塑料（Polyethylene Microplastics,PE-MPs）为对象,探究其对融合菌株F14在降解菲过程中的影响。采用扫描电镜（SEM）对添加PE-MPs前后细胞形态进行表征,结果显示,添加PE-MPs后的细胞呈现明显的凹陷,且胞外聚合物明显增多;采用傅里叶变换光谱（FTIR）对F14菌株细胞表面化学成分进行表征,发现在接触PE-MPs后,菌株细胞表面的碳水化合物、多糖和酰胺的吸收强度发生了相对变化,蛋白质、核酸、多糖的相对含量明显增加;细胞活性氧测试表明,随着PE-MPs浓度的增加和培养时间的延长,F14细胞内活性氧含量逐渐降低,PE-MPs的粒径对细胞活性氧的产生量没有明显的影响。试验浓度和粒径范围内的PE-MPs的存在并没有抑制融合菌株F14对菲的降解,反而有所促进,推测可能是PE-MPs作为载体增大了F14和菲的接触机会,同时其可能对F14菌株细胞产生了应激反应,促使F14分泌胞外聚合物,同时减弱了F14细胞的氧化损伤,进而影响了F14对菲的降解过程。     

Detecting and measuring cotranslational protein degradation in vivo

Nascent polypeptides emerging from the ribosome and not yet folded may at least transiently present degradation signals similar to those recognized by the ubiquitin system in misfolded proteins. The ubiquitin sandwich technique was used to detect and measure cotranslational protein degradation in living cells. More than 50 percent of nascent protein molecules bearing an amino-terminal degradation signal can be degraded cotranslationally, never reaching their mature size before their destruction by processive proteolysis. Thus, the folding of nascent proteins, including abnormal ones, may be in kinetic competition with pathways that target these proteins for degradation cotranslationally.     

Fish oils inhibit endothelial cell production of platelet-derived growth factor-like protein

Diets rich in fish and fish oils are associated with a reduced risk of cardiovascular disease and atherosclerosis. The interaction of a commercial fish oil extract (MaxEPA) with vascular endothelial cells (ECs) was studied as a possible mechanism for this protective effect. MaxEPA almost completely inhibited EC production of platelet-derived growth factor-like protein (PDGFc) while other lipids had a lesser effect or no effect. Overall protein synthesis was not reduced, nor was the inhibition due to defective secretion or increased degradation of the growth factor. Antioxidants suppressed the inhibitory activity of MaxEPA indicating that free radical oxidative processes were required for the inhibition. These results suggest that fish oils may suppress intimal smooth muscle cell proliferation by decreasing the production of EC-derived paracrine growth factors. This inhibitory process represents a possible molecular mechanism for the antiatherosclerotic action of marine lipids.     

Apoptotic force and tissue dynamics during Drosophila embryogenesis

Understanding cell morphogenesis during metazoan development requires knowledge of how cells and the extracellular matrix produce and respond to forces. We investigated how apoptosis, which remodels tissue by eliminating supernumerary cells, also contributes forces to a tissue (the amnioserosa) that promotes cell-sheet fusion (dorsal closure) in the Drosophila embryo. We showed that expression in the amnioserosa of proteins that suppress or enhance apoptosis slows or speeds dorsal closure, respectively. These changes correlate with the forces produced by the amnioserosa and the rate of seam formation between the cell sheets (zipping), key processes that contribute to closure. This apoptotic force is used by the embryo to drive cell-sheet movements during development, a role not classically attributed to apoptosis.     

乙嘧酚在黄瓜和土壤中的消解动态研究

利用高效液相色谱仪及田间试验方法,建立了乙嘧酚在黄瓜和土壤中的残留分析方法,研究了乙嘧酚在黄瓜和土壤中的残留消解动态,对影响残留分析方法的主要参数进行了优化.黄瓜和土壤样品分别用乙腈和丙酮提取,硅胶柱净化,高效液相色谱仪二极管阵列检测器检测,外标法定量.结果表明,该方法的最小检出量为3.5×10-10g,在黄瓜和土壤中的最低检测浓度分别为0.010和0.005 mg·kg~(-1).乙嘧酚的平均添加回收率为80.5%～103.1%,变异系数为2.10%～3.74%.消解动态试验表明,乙嘧酚的残留量随时间延长而降低,消解动态曲线符合一级动力学方程,在黄瓜和土壤中的半衰期分别为3.5和9.9 d,属于易降解性农药化合物.乙嘧酚在黄瓜中消解速率高于其在土壤中的消解速率,这可能是由于黄瓜生长稀释作用导致的.     

Road to ruin: targeting proteins for degradation in the endoplasmic reticulum

Some nascent proteins that fold within the endoplasmic reticulum (ER) never reach their native state. Misfolded proteins are removed from the folding machinery, dislocated from the ER into the cytosol, and degraded in a series of pathways collectively referred to as ER-associated degradation (ERAD). Distinct ERAD pathways centered on different E3 ubiquitin ligases survey the range of potential substrates. We now know many of the components of the ERAD machinery and pathways used to detect substrates and target them for degradation. Much less is known about the features used to identify terminally misfolded conformations and the broader role of these pathways in regulating protein half-lives.     

噻菌灵杀菌剂在两种土壤中残留降解因素分析

【目的】对比噻菌灵杀菌剂在不同土壤中的残留降解差异。【方法】研究使用高效液相色谱法分析了噻菌灵杀菌剂在两种土壤中不同温度、不同光照条件下的残留和降解动态。样品在超声振荡条件下用乙腈提取,高效液相色谱仪(配置紫外检测器)检测。添加量在5~10mg/kg。添加回收试验结果表明噻菌灵杀菌剂在两种土壤中的添加回收率为84.1%~90.2%,变异系数为0.98%~1.84%。【结果】试验结果表明,在添加5.0mg/kg和10.0mg/kg噻菌灵的土壤中,30℃条件下,在北京潮褐土中半衰期分别为23.9,24.1d,在东北黑土中的半衰期分别为18.7,21.1d,40℃时,噻菌灵在北京潮褐土和东北黑土中的降解半衰期分别为16.5,21.6d和14,18.9d。光照试验表明,在300 W高压汞灯照射下,添加10.0mg/kg时,噻菌灵在北京潮褐土和东北黑土中的半衰期分别为1.8,1.3d。【结论】此方法的灵敏度、准确度和精密度均符合农药残留测定的技术要求。     

Protection of dentate hilar cells from prolonged stimulation by intracellular calcium chelation

Prolonged afferent stimulation of the rat dentate gyrus in vivo leads to degeneration only of those cells that lack immunoreactivity for the calcium binding proteins parvalbumin and calbindin. In order to test the hypothesis that calcium binding proteins protect against the effects of prolonged stimulation, intracellular recordings were made in hippocampal slices from cells that lack immunoreactivity for calcium binding proteins. Calcium binding protein-negative cells showed electrophysiological signs of deterioration during prolonged stimulation; cells containing calcium binding protein did not. When neurons without calcium binding proteins were impaled with microelectrodes containing the calcium chelator BAPTA, and BAPTA was allowed to diffuse into the cells, these cells showed no deterioration. These results indicate that, in a complex tissue of the central nervous system, an activity-induced increase in intracellular calcium can trigger processes leading to cell deterioration, and that increasing the calcium binding capacity of a cell decreases its vulnerability to damage.     

Linking albinism and immunity: the secrets of secretory lysosomes

Lysosomes are membrane-bound organelles that are found in all mammalian cells and contain hydrolases and lipases required for protein and membrane degradation. In many cells of the immune system, lysosomes also contain secretory proteins that can be released by regulated exocytosis in response to an external stimulus, providing different cell types with a wide range of effector functions. Melanosomes also use a lysosome-related organelle to secrete melanin for pigmentation. Links between albinism and immunity in patients have uncovered a number of key proteins required for lysosomal secretion and have revealed a versatile secretory mechanism that can be fine-tuned by distinct interactions in different cell types.     

木醋液对土壤pH、EC与茄子叶片光合特性及根系发育的影响

为探究木醋液对土壤性质和植物生长发育的影响,以玉米、小麦秸秆和杂木在制备生物炭过程中产生的木醋液为研究材料,通过盆栽试验,研究了灌施不同稀释倍数的木醋液对土壤pH、EC（电导率）和茄子生长发育的影响。结果表明,与灌施清水相比,土壤pH值随着木醋液稀释倍数的增大而增大,当稀释倍数增大到1∶300时pH值与灌施清水处理无明显差异。灌施木醋液后土壤EC值随着木醋液稀释倍数的增大而减小,当稀释倍数增大到1∶300时EC值与灌施清水处理差异不显著。生物试验表明,灌施稀释倍数小于10倍的木醋液会对茄子产生毒害作用,灌施1∶50以上的木醋液可以增强茄子叶片净光合作用,增加叶绿素含量,促进茄子的生长发育和根系生长。木醋液对作物的促进作用随着灌施木醋液稀释倍数的增加逐渐减弱,灌施稀释倍数1∶300的木醋液与灌施清水处理差异不显著。研究表明,浇灌适宜稀释倍数的木醋液可以调节土壤pH和EC值,促进茄子生长,这为木醋液在蔬菜种植中的应用推广提供了参考。     

Synaptic protein degradation underlies destabilization of retrieved fear memory

Reactivated memory undergoes a rebuilding process that depends on de novo protein synthesis. This suggests that retrieval is dynamic and serves to incorporate new information into preexisting memories. However, little is known about whether or not protein degradation is involved in the reorganization of retrieved memory. We found that postsynaptic proteins were degraded in the hippocampus by polyubiquitination after retrieval of contextual fear memory. Moreover, the infusion of proteasome inhibitor into the CA1 region immediately after retrieval prevented anisomycin-induced memory impairment, as well as the extinction of fear memory. This suggests that ubiquitin- and proteasome-dependent protein degradation underlies destabilization processes after fear memory retrieval. It also provides strong evidence for the existence of reorganization processes whereby preexisting memory is disrupted by protein degradation, and updated memory is reconsolidated by protein synthesis.     

Nicotinic antagonists enhance process outgrowth by rat retinal ganglion cells in culture

Functional nicotinic cholinergic receptors are found on mammalian retinal ganglion cell neurons in culture. The neurotransmitter acetylcholine (ACh) can be detected in the medium of many of these retinal cultures, after release presumably from the choline acetyltransferase-positive amacrine cells. The postsynaptic effect of endogenous or applied ACh on the ganglion cells can be blocked with specific nicotinic antagonists. Here it is shown that within 24 hours of producing such a pharmacologic blockade, the retinal ganglion cells begin to sprout or regenerate neuronal processes. Thus, the growth-enhancing effect of nicotinic antagonists may be due to the removal of inhibition to growth by tonic levels of ACh present in the culture medium. Since there is a spontaneous leak of ACh in the intact retina, the effects of nicotinic cholinergic drugs on process outgrowth in culture may reflect a normal control mechanism for growth or regeneration of retinal ganglion cell processes that is exerted by ACh in vivo.     

Development and use of fluorescent protein markers in living cells

The ability to visualize, track, and quantify molecules and events in living cells with high spatial and temporal resolution is essential for understanding biological systems. Only recently has it become feasible to carry out these tasks due to the advent of fluorescent protein technology. Here, we trace the development of highly visible and minimally perturbing fluorescent proteins that, together with updated fluorescent imaging techniques, are providing unprecedented insights into the movement of proteins and their interactions with cellular components in living cells.