柽柳bZIP基因调控耐盐相关基因的表达

本研究探讨了柽柳（Tamarixhispida）bZIP（basicleucine zipper）基因对抗逆基因表达的调控。我们比较了非盐胁迫和盐胁迫条件下非转基因和转bZIP基因植株的SOD、POD、ATPase、GST、LTP和LEA等基因表达量的变化。结果表明,在非胁迫条件下,bZIP转录因子可能直接调控了poxN1、TOBPXD、TOBLTP和ltp1基因的表达,而其他基因的表达可能不受bZIP转录因子的直接调控。在盐胁迫下,bZIP转录因子能够直接或间接地调控部分抗逆基因的表达,使它们的表达量显著增强。     

Characterization of the TGN exit routes in AtT20 cells using pancreatic amylase and serum albumin

The AtT20 pituitary cell is the one that was originally used to define the pathways taken by secretory proteins in mammalian cells. It possesses two secretory pathways, the constitutive for immediate secretion and the regulated for accumulation and release under hormonal stimulation. It is in the regulated pathway, most precisely in the immature granule of the regulated pathway, that proteolytic maturation takes place. A pathway that stems from the regulated one, namely the constitutive-like pathway releases proteins present in immature granules that are not destined for accumulation in mature granules. In AtT20 cells proopiomelanocortin the endogenous precursor of the accumulated adrenocorticotropic hormone, is predominantly secreted in a constitutive manner without proteolytic maturation. In order to better understand by which secretory pathway intact proopiomelanocortin is secreted by a cell line possessing a regulated secretory pathway, it was transfected with rat serum albumin (a marker of constitutive secretory proteins), and pancreatic amylase (a marker of regulated proteins). COS cells were also transfected in order to serve as control of release by the constitutive pathway. It was observed that both the basal and stimulated secretions of albumin and proopiomelanocortin from AtT20 cells are identical. In addition, secretagogue stimulation when POMC is in transit in the trans-Golgi network decreases its constitutive secretion by 50%. It was also observed using cell fractionation and 20 degrees C secretion blocks that albumin and proopiomelanocortin are present in the regulated pathway, presumably in the immature granules, and are secreted by the constitutive-like secretory pathway. These observations show that stimulation can increase sorting into the regulated pathway, and confirm the importance of the constitutive-like secretory pathway in the model AtT20 cell line.     

31P-NMR study of the regulation of glycogenolysis in living skeletal muscle

Based on in vitro biochemical experiments, it is generally believed that glycogenolysis is regulated in two different ways; i.e., Ca2+ regulation at the phosphorylase step and phosphate-product regulation at the phosphofructokinase step. Recent studies on glycogenolysis in living vertebrate skeletal muscles using 31P nuclear magnetic resonance (NMR) presented evidence that glycogenolysis in vivo is regulated by Ca2+ released from the sarcoplasmic reticulum. We performed 31P-NMR studies on living frog skeletal muscle, and found that glycogenolysis is further regulated by the accumulation of phosphate products by contractile activity. Therefore, glycogenolysis in vivo can actually be regulated by the two mechanisms as predicted by in vitro biochemical studies.     

调亏灌溉和生物炭对大豆生长、产量及水分利用效率的影响

为了解生物炭及调亏灌溉对大豆的影响,以大豆"开育12号"为试验材料,采用随机区组试验设计,利用盆栽栽培条件,研究不同生物炭添加量B₀(0 t·hm^-2)、B₁(6t·hm^-2)、B₂(12 t·hm^-2)和不同程度调亏灌溉W₁(充分灌溉,70%田间持水量)、W₂(轻度调亏,55%～60%田间持水量)、W₃(重度调亏,45%～50%田间持水量)对大豆生长、产量及水分利用的影响。结果表明:轻度调亏灌溉不会影响大豆叶面积指数及地下部分干物质累积量,而大豆叶面积指数和地下部分干物质累积量随着生物炭使用量的增加而增加;地上部分干物质累积量随着调亏程度的加重而降低,而生物炭施用量为12 t·hm-2时,才会提高地上部分干物质累积量。调亏灌溉和生物炭均能影响大豆的耗水量,其中耗水量随着调亏程度的加剧而减少,而添加6 t·hm^-2生物炭耗水量最高,但有利于产量的形成。与充分灌溉不施用生...     

A review of terms for regulated vs. forced,neurochemical-induced changes in body temperature

Deviations of the body temperature of homeothermic animals may be regulated or forced. A regulated change in core temperature is caused by a natural or synthetic compound that displaces the set-point temperature. A forced shift occurs when an excessive environmental or endogenous heat load, or heat sink, exceeds the body's capacity to thermoregulate but does not affect set-point. A fever is the paradigm of a regulated increase in body temperature, but the term fever has acquired a strict pathological definition over the past two decades. Consequently, other forms of nonpathological, regulated elevations in body temperature have generally been classified as hyperthermia; and decreases in core temperature--either forced or regulated--have generally been classified as hypothermia. Since the terms hyperthermia and hypothermia fail to distinguish a regulated vs. a forced temperature change, a confusion of terms has been created in the literature. It would appear that “resisted or unregulated hyperthermia” and “hypothermia,” respectively, are appropriate terms for describing a forced increase and decrease in core temperature. A nonpathological but regulated elevation in temperature may be defined as unresisted or regulated hyperthermia, whereas a regulated decrease in temperature may be termed unresisted or regulated hypothermia. This simple scheme appears to be the most practical means for distinguishing between forced and regulated changes in core temperature.     

Rad23 and Rpn10: perennial wallflowers join the melee

Substrate ubiquitination is highly regulated; by contrast, substrate targeting to the proteasome has been considered a stochastic process that is mediated primarily by high-affinity interaction with multi-ubiquitin chains. However, recent findings have shown that substrate recognition by the proteasome is also regulated, and requires Rad23, Dsk2 and Rpn10. These studies suggest that the engagement of protein ubiquitination and translocation with degradation by the proteasome is coordinated by a series of regulated events.     

Effect of skeletal muscle myosin light chain 2 on the Ca2+-sensitive interaction of myosin and heavy meromyosin with regulated actin

A low-speed centrifugation assay has been used to examine the binding of myosin filaments to F-action and to regulated actin in the presence of MgATP. While the cross-linking of F-actin by myosin was Ca2+ insensitive, much less regulated actin was cross-linked by myosin in the absence of Ca2+ than in its presence. Removal of the 19000-dalton, phosphorylatable light chain from myosin resulted in the loss of this Ca2+ sensitivity. Readdition of this light chain partially restored the Ca2+-sensitive cross-linking of regulated actin by myosin. Urea gel electrophoresis has been used to distinguish that fraction of heavy meromyosin which contains intact phosphorylatable light chain from that which contains a 17000-dalton fragment of this light chain. In the absence of Ca2+, heavy meromyosin which contained digested light chain bound to regulated actin in MgATP about 10-fold more tightly than did heavy meromyosin which contained intact light chain. The regulated actin-activated ATPases of heavy meromyosin also showed that cleavage of this light chain causes a substantial increase in the affinity of heavy meromyosin for regulated actin in the absence of Ca2+. Thus, the binding of both myosin and heavy meromyosin to regulated actin is Ca2+ sensitive, and this sensitivity is dependent on the phosphorylatable light chain.     

Intracellular organelle transport: few motors, many signals

Bidirectional microtubule-dependent organelle transport in melanophores is regulated by cAMP through organelle-bound protein kinase A (PKA); however, the mechanisms responsible for this regulation are unknown. A recent study by Gelfand and colleagues demonstrates that, in addition to PKA, transport is regulated by the organelle-bound mitogen-activated protein kinase (MAPK) signaling components ERK and MEK, whose activity is required for bidirectional transport along microtubules. This pathway apparently acts downstream of PKA, suggesting that bidirectional organelle transport is regulated by a hierarchical cascade of signaling pathways.     

Spatial segregation of the regulated and constitutive secretory pathways

Recent experiments using DNA transfection have shown that secretory proteins in AtT-20 cells are sorted into two biochemically distinct secretory pathways. These two pathways differ in the temporal regulation of exocytosis. Proteins secreted by the regulated pathway are stored in dense-core granules until release is stimulated by secretagogues. In contrast, proteins secreted by the constitutive pathway are exported continuously, without storage. It is not known whether there are mechanisms to segregate regulated and constitutive secretory vesicles spatially. In this study, we examined the site of insertion of constitutive vesicles and compared it with that of regulated secretory granules. Regulated granules accumulate at tips of processes in these cells. To determine whether constitutively externalized membrane proteins are inserted into plasma membrane at the cell body or at process tips, AtT-20 cells were infected with ts-O45, a temperature-sensitive mutant of vesicular stomatitis virus in which transport of the surface glycoprotein G is conditionally blocked in the ER. After switching to the permissive temperature, insertion of G protein was detected at the cell body, not at process tips. Targeting of constitutive and regulated secretory vesicles to distinct areas of the plasma membrane appears to be mediated by microtubules. We found that while disruption of microtubules by colchicine had no effect on constitutive secretion, it completely blocked the accumulation of regulated granules at special release sites. Colchicine also affected the proper packaging of regulated secretory proteins. We conclude that regulated and constitutive secretory vesicles are targeted to different areas of the plasma membrane, most probably by differential interactions with microtubules. These results imply that regulated secretory granules may have unique membrane receptors for selective attachment to microtubules.     

Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease

The regulated secretory pathway of neurons is the major source of extracellular A beta that accumulates in Alzheimer's disease (AD). Extracellular A beta secreted from that pathway is generated by beta-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with A beta in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to A beta in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular A beta released by the regulated secretory pathway, but CA074Me had no effect on A beta released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal beta-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate beta-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as beta-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce A beta in AD.     

茶丽纹象甲为害诱导的茶树防御反应分析

基于转录组测序,获得了茶树新梢叶片被茶丽纹象甲为害前后的差异基因表达谱,并从信号监测与转导、转录因子、次生代谢物生成等方面分析了相关基因的表达变化情况,为进一步研究茶丽纹象甲为害诱导的茶树防御机制奠定基础。结果表明:(1)茶丽纹象甲为害茶树新梢叶片后共检测到显著上调表达基因309个,显著下调表达基因297个,这些显著差异表达基因共分成23类。(2)检测到信号监测与转导过程中合计17个单基因上调表达2.0～2.8倍,包括富含亮氨酸重复序列的类受体蛋白激酶基因、促分裂原活化蛋白激酶级联信号系统蛋白激酶基因、茉莉酸信号通路基因、钙离子信号基因、水杨酸代谢通路基因;检测到21个转录因子单基因上调表达1.8～2.8倍,包括bHLH、WRKY、bZIP、MYB、UAF等转录因子;检测到苯丙类、类黄酮及萜类物质代谢过程中合计8个单基因上调表达2.1～2.5倍,包括苯丙酮酸互变异构酶基因、肉桂酰辅酶A还原酶基因、二氢黄酮醇还原酶基因、花色素还原酶基因、法尼基二磷酸合成酶基因、法尼醇脱氢酶基因。研究认为,茶丽纹象甲为害诱导信号转导、转录因子及次生代谢过程中的基因增量表达以机械损伤作用为主,昆虫口腔分泌物对这些基因的诱导表达具有协同促进作用。     

Regulation of extranuclear PtdIns5P production by phosphatidylinositol phosphate 4-kinase 2alpha

Cellular levels of the phosphoinositide PtdIns5P are regulated by agonist stimulation, but the mechanisms controlling turnover of this lipid, and the subcellular location of the regulated PtdIns5P pool(s), remain poorly understood. Here we show that enhanced tyrosine phosphorylation robustly increases cellular PtdIns5P levels. Moreover, unlike PtdIns5P production enhanced by cell stress, we show that this pool of PtdIns5P is specifically regulated by the inositol lipid kinase PIP4K2a.     

Regulated and constitutive secretion. Differential effects of protein synthesis arrest on transport of glycosaminoglycan chains to the two secretory pathways.

Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et al. (Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) Cell 50, 289-300) have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be glycosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, suggesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged along with the hormone adrenocorticotropin (ACTH). We found that cycloheximide treatment severely impairs the cell's ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.     

Use of bacteriocin promoters for gene expression in Lactobacillus plantarum C11

AIMS: To exploit promoters involved in production of the bacteriocin sakacin P for regulated overexpression of genes in Lactobacillus plantarum C11. METHODS AND RESULTS: Production of sakacin P by Lact. sakei LTH673 is controlled by a peptide-based quorum sensing system that drives strong, regulated promoters. One of these promoters (PorfX) was used to establish regulated overexpression of genes encoding chloramphenicol acetyltransferase from Bacillus pumilus, aminopeptidase N from Lactococcus lactis or chitinase B from Serratia marcescens in Lact. plantarum C11, a strain that naturally possesses the regulatory machinery that is necessary for promoter activation. The expression levels obtained were highly dependent on which gene was used and on how the promoter was coupled to this gene. The highest expression levels (14% of total cellular protein) were obtained with the aminopeptidase N gene translationally fused to the regulated promoter. CONCLUSIONS: Sakacin promoters permit regulated expression of a variety of genes in Lact. plantarum C11. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the usefulness of regulated bacteriocin promoters for developing new gene expression systems for lactic acid bacteria, in particular lactobacilli.