A human TERT C-terminal polypeptide sensitizes HeLa cells to H2O2-induced senescence without affecting telomerase enzymatic activity

We have constructed a 27-kDa hTERT C-terminal polypeptide (hTERTC27) devoid of domains required for telomerase activity and demonstrated that it is capable of nuclear translocation/telomere-end targeting. Here we showed that expression of a low level of hTERTC27 renders hTERT positive HeLa cells sensitive to H(2)O(2)-induced oxidative stress and subsequent cell senescence. The senescence-associated gene, the cyclin/cdk inhibitor p21(Waf1), was up-regulated. This occurs without changing the expression of endogenous hTERT, causing significant telomere shortening or inhibiting telomerase activity. Results from this study suggest for the first time that in addition to telomerase activity, the C-terminus of hTERT also plays a role in hTERT-mediated cellular resistance to oxidative stress.     

台湾相思叶状柄衰老过程中氮磷内吸收变化研究

对厦门五老峰台湾相思成熟和衰老叶状柄的N、P含量、N:P比值及内吸收率的研究表明:(1)台湾相思成熟叶状柄具有高的N含量,冬、春季分别为39.90±2.10 mg/g、43.13±1.67 mg/g,P含量冬、春季分别为1.24±0.04 mg/g、1.16±0.05 mg/g,叶状柄在衰老过程中,N、P含量明显下降;(2)成熟叶状柄的N:P比值冬、春季分别为32.26±1.53、37.37±1.61,均高于16,显示厦门五老峰台湾相思林存在P限制,但叶状柄衰老过程中P的内吸收率显著高于N的内吸收率。     

Astrocyte Leucine Metabolism: Significance of Branched-Chain Amino Acid Transamination

Abstract: We studied astrocytic metabolism of leucine, which in brain is a major donor of nitrogen for the synthesis of glutamate and glutamine. The uptake of leucine into glia was rapid, with a V _max of 53.6 ± 3.2 nmol/mg of protein/min and a K _m of 449.2 ± 94.9 µ M . Virtually all leucine transport was found to be Na⁺ independent. Astrocytic accumulation of leucine was much greater (3×) in the presence of α-aminooxyacetic acid (5 m M ), an inhibitor of transamination reactions, suggesting that the glia rapidly transaminate leucine to α-ketoisocaproic acid (KIC), which they then release into the extracellular fluid. This inference was confirmed by the direct measurement of KIC release to the medium when astrocytes were incubated with leucine. Approximately 70% of the leucine that the glia cleared from the medium was released as the keto acid. The apparent K _m for leucine conversion to extracellular KIC was a medium [leucine] of 58 µ M with a V _max of ∼2.0 nmol/mg of protein/min. The transamination of leucine is bidirectional (leucine + α-ketoglutarate ↮ KIC + glutamate) in astrocytes, but flux from leucine → glutamate is more active than that from glutamate → leucine. These data underscore the significance of leucine handling to overall brain nitrogen metabolism. The release of KIC from glia to the extracellular fluid may afford a mechanism for the "buffering" of glutamate in neurons, which would consume this neurotransmitter in the course of reaminating KIC to leucine.     

The N Terminus of Pro-endothelial Monocyte-activating Polypeptide II (EMAP II) Regulates Its Binding with the C Terminus,Arginyl-tRNA Synthetase,and Neurofilament Light Protein

Pro-endothelial monocyte-activating polypeptide II (EMAP II), one component of the multi-aminoacyl tRNA synthetase complex, plays multiple roles in physiological and pathological processes of protein translation, signal transduction, immunity, lung development, and tumor growth. Recent studies have determined that pro-EMAP II has an essential role in maintaining axon integrity in central and peripheral neural systems where deletion of the C terminus of pro-EMAP II has been reported in a consanguineous Israeli Bedouin kindred suffering from Pelizaeus-Merzbacher-like disease. We hypothesized that the N terminus of pro-EMAP II has an important role in the regulation of protein-protein interactions. Using a GFP reporter system, we defined a putative leucine zipper in the N terminus of human pro-EMAP II protein (amino acid residues 1–70) that can form specific strip-like punctate structures. Through GFP punctum analysis, we uncovered that the pro-EMAP II C terminus (amino acids 147–312) can repress GFP punctum formation. Pulldown assays confirmed that the binding between the pro-EMAP II N terminus and its C terminus is mediated by a putative leucine zipper. Furthermore, the pro-EMAP II 1–70 amino acid region was identified as the binding partner of arginyl-tRNA synthetase, a polypeptide of the multi-aminoacyl tRNA synthetase complex. We also determined that the punctate GFP pro-EMAP II 1–70 amino acid aggregate colocalizes and binds to the neurofilament light subunit protein that is associated with pathologic neurofilament network disorganization and degeneration of motor neurons. These findings indicate the structure and binding interaction of pro-EMAP II protein and suggest a role of this protein in pathological neurodegenerative diseases.     

A Role for Glutamine Synthetase in the Remobilization of Leaf Nitrogen during Natural Senescence in Rice Leaves

Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of corresponding mRNAs were determined in leaves of hydroponically grown rice (Oryza sativa) plants during natural senescence. The plants were grown in the greenhouse for 105 days at which time the thirteenth leaf was fully expanded. This was counted as zero time for senescence of the twelfth leaf. The twelfth leaf blade on the main stem was analyzed over a time period of −7 days (98 days after germination) to +42 days (147 days after germination). Total GS activity declined to less than a quarter of its initial level during the senescence for 35 days and this decline was mainly caused by a decrease in the amount of GS2 polypeptide. Immunoblotting analyses showed that contents of other chloroplastic enzymes, such as ribulose-1,5-bisphosphate carboxylase/oxygenase and Fd-glutamate synthase, declined in parallel with GS2. In contrast, the GS1 polypeptide remained constant throughout the senescence period. Translatable mRNA for GS1 increased about fourfold during the senescence for 35 days. During senescence, there was a marked decrease in content of glutamate (to about one-sixth of the zero time value); glutamate is the major form of free amino acid in rice leaves. Glutamine, the major transported amino acid, increased about threefold compared to the early phase of the harvest in the senescing rice leaf blades. These observations suggest that GS1 in senescing leaf blades is responsible for the synthesis of glutamine, which is then transferred to the growing tissues in rice plants.     

Development of leucine aminopeptidase activity during daylily flower growth and senescence

The possibility that exopeptidases, i.e. aminopeptidases and carboxypeptidases, in addition to the previously studied endopeptidase might also be developmentally regulated in daylily petals was examined. The level of leucine aminopeptidase and endopeptidase activities changed after the flower was fully open while that of carboxypeptidase activity remained relatively unchanged throughout senescence. Leucine aminopeptidase activity seemed to increase after the flower was fully open and peaked several hours earlier than endopeptidase did. Taken together, it is postulated that leucine aminopeptidase might play a role in protein turnover during flower opening and in the initiation of protein hydrolysis associated with petal senescence while the endopeptidase could be responsible for the breakdown of the bulk of proteins at the later stages. The drop in leucine aminopeptidase activity associated with the onset of daylily petal senescence was effectively halted by a cycloheximide treatment of cut daylily flowers for 24 h which was previously shown to prolong the vase life of the flowers and prevent protein loss from the petals. Apart from both being developmentally regulated in daylily petals, the leucine aminopeptidase activity and the previously studied endopeptidase are different in several aspects. They appear to have different pH optima, 8 for leucine aminopeptidase and 6.2 for endopeptidase. Unlike the endopeptidase activity, no new leucine aminopeptidase isozymes appeared during petal senescence, and the leucine aminopeptidase did not appear to belong to the cysteine class of proteolytic enzymes.     

The trimerization domain of NEMO is composed of the interacting C-terminal CC2 and LZ coiled-coil subdomains

NEMO (NF-kappaB essential modulator) plays a key role in the canonical NF-kappaB pathway as the scaffold/regulatory component of the IkappaB kinase (IKK) complex. The self-association of NEMO involves the C-terminal halves of the polypeptide chains containing two putative coiled-coil motifs (a CC2 and a LZ leucine zipper), a proline-rich region, and a ZF zinc finger motif. Using purified truncation mutants, we showed that the minimal oligomerization domain of NEMO is the CC2-LZ segment and that both CC2 and LZ subdomains are necessary to restore the LPS-dependent activation of the NF-kappaB pathway in a NEMO-deficient cell line. We confirmed the association of the oligomerization domain in a trimer and investigated the specific role of CC2 and LZ subdomains in the building of the oligomer. Whereas a recombinant CC2-LZ polypeptide self-associated into a trimer with an association constant close to that of the wild-type protein, the isolated CC2 and LZ peptides, respectively, formed trimers and dimers with weaker association constants. Upon mixing, isolated CC2 and LZ peptides associated to form a stable hetero-hexamer as shown by gel filtration and fluorescence anisotropy experiments. We propose a structural model for the organization of the oligomerization domain of activated NEMO in which three C-terminal domains associate into a pseudo-hexamer forming a six-helix bundle. This model is discussed in relation to the mechanism of activation of the IKK complex by upstream activators.     

Nogo-B抑制Ox-LDL诱导的巨噬细胞泡沫化

Nogo-B在血管损伤、组织修复和炎症反应中发挥重要作用。然而,Nogo-B在动脉粥样硬化中的作用仍不明确。本研究拟在巨噬细胞中探讨Nogo-B对巨噬细胞泡沫化的影响。在RAW264.7细胞中沉默Nogo-B后,采用氧化低密度脂蛋白(Ox-LDL)或DiI修饰的Ox-LDL诱导巨噬细胞泡沫化;通过激光共聚焦显微镜观察巨噬细胞中荧光脂质,并在透射电镜下观察各组细胞中自噬泡;采用Western 印迹分析Plin2、p62和LC3-II的蛋白质水平;采用实时荧光定量PCR检测p62 mRNA水平;采用氯喹处理以及mRFP-GFP-LC3双荧光体系分析自噬流功能;进一步过表达Nogo-B后,比较巨噬细胞中脂质负荷程度以及Plin2、p62和LC3-II的蛋白质水平。结果显示,DiI-Ox-LDL处理后,Nogo-B沉默组细胞中脂质负荷程度高于对照组（2.34±0.67 vs. 0.69±0.14,P＜0.05）;Ox-LDL处理后,Nogo-B沉默组细胞中自噬泡数量（8.67±0.58 vs. 4.33±0.58,P＜0.01）、Plin2（4.65±0.50 vs. 3.24±0.71,P＜0.05）、p62（10.13±1.79 vs. 5.76±1.84,P＜0.05）和LC3-II（4.38±0.20 vs. 2-33±1.56,P＜0.01）的蛋白质水平均显著高于对照组,而p62 mRNA水平无差异（P＞0.05）;进一步研究发现,Nogo-B沉默组的自噬流被抑制了;过表达Nogo-B后,虽然p62蛋白质水平无明显变化,但是细胞中脂质负荷程度显著低于对照组（1.68±1.06 vs. 4.94±0.70,P＜0.05）,Plin2和LC3-II的蛋白质水平也明显降低。上述结果表明,Nogo-B通过促进自噬流抑制了Ox-LDL诱导的巨噬细胞泡沫化,Nogo-B可能具有抗动脉粥样硬化的作用。     

Nogo-B抑制Ox-LDL诱导的巨噬细胞泡沫化

Nogo-B在血管损伤、组织修复和炎症反应中发挥重要作用。然而,Nogo-B在动脉粥样硬化中的作用仍不明确。本研究拟在巨噬细胞中探讨Nogo-B对巨噬细胞泡沫化的影响。在RAW264.7细胞中沉默Nogo-B后,采用氧化低密度脂蛋白(Ox-LDL)或DiI修饰的Ox-LDL诱导巨噬细胞泡沫化;通过激光共聚焦显微镜观察巨噬细胞中荧光脂质,并在透射电镜下观察各组细胞中自噬泡;采用Western 印迹分析Plin2、p62和LC3-II的蛋白质水平;采用实时荧光定量PCR检测p62 mRNA水平;采用氯喹处理以及mRFP-GFP-LC3双荧光体系分析自噬流功能;进一步过表达Nogo-B后,比较巨噬细胞中脂质负荷程度以及Plin2、p62和LC3-II的蛋白质水平。结果显示,DiI-Ox-LDL处理后,Nogo-B沉默组细胞中脂质负荷程度高于对照组（2.34±0.67 vs. 0.69±0.14,P＜0.05）;Ox-LDL处理后,Nogo-B沉默组细胞中自噬泡数量（8.67±0.58 vs. 4.33±0.58,P＜0.01）、Plin2（4.65±0.50 vs. 3.24±0.71,P＜0.05）、p62（10.13±1.79 vs. 5.76±1.84,P＜0.05）和LC3-II（4.38±0.20 vs. 2-33±1.56,P＜0.01）的蛋白质水平均显著高于对照组,而p62 mRNA水平无差异（P＞0.05）;进一步研究发现,Nogo-B沉默组的自噬流被抑制了;过表达Nogo-B后,虽然p62蛋白质水平无明显变化,但是细胞中脂质负荷程度显著低于对照组（1.68±1.06 vs. 4.94±0.70,P＜0.05）,Plin2和LC3-II的蛋白质水平也明显降低。上述结果表明,Nogo-B通过促进自噬流抑制了Ox-LDL诱导的巨噬细胞泡沫化,Nogo-B可能具有抗动脉粥样硬化的作用。     

Asparagine and regulation of photoinduced rhythm in Penicillium claviforme

A photosensitive reaction involved in the expression and regulation of the endogenous sporulation rhythm in Penicillium claviforme Bainier CBS 126-23 has been described earlier by our team. The present work shows that asparagine plays a central role in this periodic system. The 24–26 h periodicity, which becomes desynchronized over a few days, was affected by supplying asparagine (1 to 25 m M ). a) The rhythm remained synchronized for at least 3 weeks, for all the asparagine concentrations used, b) The period increased successively from one cycle to the next (from 35±3 h to 73±8 h over 6 cycles) for 1 m M asparagine. This period lengthening became less and less accentuated as the asparagine concentration was increased. At ≥10 m M , the period was stable; it was 33±5 h for 10 m M and 22±1.5 h for 25 m M asparagine. Otherwise asparagine could not elicit rhythmicity in darkness or in dim continuous light (≤5 μW m⁻²).     

Leucine stimulates insulin secretion via down-regulation of surface expression of adrenergic α2A receptor through the mTOR (mammalian target of rapamycin) pathway: implication in new-onset diabetes in renal transplantation

The amino acid leucine is a potent secretagogue, capable of inducing insulin secretion. It also plays an important role in the regulation of mTOR activity, therefore, providing impetus to investigate if a leucine-sensing mechanism in the mTOR pathway is involved in insulin secretion. We found that leucine-induced insulin secretion was inhibited by both the mTOR inhibitor rapamycin as well as the adrenergic α2 receptor agonist clonidine. We also demonstrated that leucine down-regulated the surface expression of adrenergic α2A receptor via activation of the mTOR pathway. The leucine stimulatory effect on insulin secretion was attenuated in diabetic Goto-Kakizaki rats that overexpress adrenergic α2A receptors, confirming the role of leucine in insulin secretion. Thus, our data demonstrate that leucine regulates insulin secretion by modulating adrenergic α2 receptors through the mTOR pathway. The role of the mTOR pathway in metabolic homeostasis led us to a second important finding in this study; retrospective analysis of clinical data showed that co-administration of rapamycin and clonidine was associated with an increased incidence of new-onset diabetes in renal transplantation patients over those receiving rapamycin alone. We believe that inhibition of mTOR by rapamycin along with activation of adrenergic α2 receptors by clonidine represents a double-hit to pancreatic islets that synergistically disturbs glucose homeostasis. This new insight may have important implications for the clinical management of renal transplant patients.     

Apple BT2 protein negatively regulates jasmonic acid‐triggered leaf senescence by modulating the stability of MYC2 and JAZ2

Jasmonic acid (JA) is shown to induce leaf senescence. However, the underlying molecular mechanism is not well understood, especially in woody plants such as fruit trees. In this study, we are interested in exploring the biological role of MdBT2 in JA‐mediated leaf senescence. We found that MdBT2 played an antagonistic role in MdMYC2‐promoted leaf senescence. Our results revealed that MdBT2 interacted with MdMYC2 and accelerated its ubiquitination degradation, thus negatively regulated MdMYC2‐promoted leaf senescence. In addition, MdBT2 acted as a stabilizing factor to improve the stability of MdJAZ2 through direct interaction, thereby inhibited JA‐mediated leaf senescence. Furthermore, our results also showed that MdBT2 interacted with a subset of JAZ proteins in apple, including MdJAZ1, MdJAZ3, MdJAZ4 and MdJAZ8. Our investigations provide new insight into molecular mechanisms of JA‐modulated leaf senescence. The dynamic JA‐MdBT2‐MdJAZ2‐MdMYC2 regulatory module plays an important role in JA‐modulated leaf senescence.     

Rate of Glutamate Synthesis from Leucine in Rat Brain Measured In Vivo by 15N NMR

Abstract: The rate of glutamate synthesis from leucine by the branched-chain aminotransferase was measured in rat brain in vivo at steady state. The rats were fed exclusively by intravenous infusion of a nutrient solution containing [¹⁵N]leucine. The rate of glutamate synthesis from leucine, determined from the rate of increase of brain [¹⁵N]glutamate measured by ¹⁵N NMR and the ¹⁵N enrichments of brain and blood leucine analyzed by gas chromatography-mass spectrometry, was 0.7–1.8 µmol/g/h at a steady-state brain leucine concentration of 0.25 µmol/g. A comparison of the observed fractional ¹⁵N enrichments of brain leucine (0.42 ± 0.03) and glutamate (0.21 ± 0.015) showed that leucine provides ∼50% of glutamate nitrogen under our experimental condition. From the observed rate (0.7–1.8 µmol/g) and the known K _m of the branched-chain aminotransferase for leucine (1.2 m M ), the rate of glutamate synthesis from leucine at physiological brain leucine concentration (0.11 µmol/g) was estimated to be 0.35–0.9 µmol/g/h, with leucine providing ∼25% of glutamate nitrogen. The results strongly suggest that plasma leucine from dietary source, transported into the brain, is an important external source of nitrogen for replenishment of brain glutamate in vivo. Implications of the results for treatment of maple-syrup urine disease patients with leucine-restricted diet are discussed.     

Storage and remobilisation of sulphur in beech trees (Fagus sylvatica)

Three-year-old beech trees were fed ³⁵S-sulphate in August 1993 via a flap in a mature leaf of an upper branch. Harvest of beech trees was performed 24 h after feeding ³⁵S-sulphate, before leaf senescence, after leaf abscission, in early winter (January 1994). in late winter (March 1994). before bud break and after bud break. Twenty-four h after feeding ³⁵S-sulphate, 0.7 ± 0.5% of the ³⁵S-radioactivity taken up was exported out of the fed leaf. When trees were analysed 2 months later, i.e., before leaf senescence, this value had increased to 22 ± 7%. The exported ³⁵S-radioactivity was located in the branch containing the fed leaf (2.8 ± 13%). in basipetal parts of the trunk (41 ± 77%) and in the main rool (21 ± 6%). Leaves and apical parts of the trunk were no sink organs for the exported sulphur. Along the tree axis the main proportion of the radiolabel was located in the wood, predominantly in the acid soluble fraction. In the bark the greater portion of the radiolabel was found in the acid insoluble fraction. In both tissues the bulk of the ³⁵S of the soluble fraction was sulphate together with small amounts of glutathione. This pattern did not change until bud break. After bud break, basipetal parts of the trunk lost part of its ³⁵S-radioactivity. Of the ³⁵S-radioactivity which had been exported out of the fed leaf during the previous autumn, 16 ± 2% remained in the trunk, whereas 47 ± 7% of the ³⁵S was found in branches, mainly in the newly developed leaves. The present results show that sulphur, mainly in the form of sulphate, is stored along the tree axis in both bark and wood of beech trees and is re-mobilised during leaf development in spring.     

Leucine promotes leptin receptor expression in mouse C2C12 myotubes through the mTOR pathway

Leptin plays a critical role in regulating muscle protein metabolism by binding with leptin receptors in a 1:1 stoichiometry. However, the role for leucine in the regulation of leptin receptor expression in muscle has not been investigated. The present study was conducted to test the hypothesis that leucine regulates leptin receptor levels in C2C12 myotubes. Cells were cultured in the presence of DMEM/F12 medium containing supplemental 0 or 5 mM l-leucine. Leptin receptor expression by C2C12 myotubes peaked at 2 h post-supplementation. Additionally, leucine stimulated leptin receptor expression at both mRNA and protein levels in a dose-dependent manner. Furthermore, leucine enhanced the phosphorylation of mammalian target of rapamycin (mTOR). Addition of rapamycin (an inhibitor of mTOR) to culture medium completely suppressed leucine-induced activation of mTOR and inhibited leucine-stimulated leptin receptor production. These results indicate that leucine affects leptin receptor expression in muscle cells via the mTOR signaling pathway.     

Synthesis of proteins by chloroplasts from iron-deficient Euglena gracilis

Chloroplasts isolated from Euglena gracilis made iron deficient by growth on 0.5 μm iron show distinct qualitative and quantitative changes in their polypeptide composition in comparison with iron-sufficient (40 μm) chloroplasts. These changes were noted in the stromal, thylakoid, and envelope subfractions. Iron-deficient chloroplasts have a sedimentation behavior similar to that of iron-sufficient chloroplasts and also contain substantial amounts of ribulose-1,5-bisphosphate carboxylase. In addition, iron-deficient chloroplasts incorporate [³H]leucine into polypeptides at rates about one-third of those from control chloroplasts (40 μm Fe) on a per-microgram-chlorophyll basis. Incorporation of [³H]leucine into specific polypeptides, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows relatively normal synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase and two of the three major chloroplast-derived polypeptides of the thylakoids. No incorporation was detected, however, into a polypeptide of ca. 33 kd which is synthesized by normal plastids. Iron-deficient chloroplasts also synthesize a stromal polypeptide of ca. 85 kd not seen in chloroplasts from normal cells. This evidence is consistent with a direct or indirect role for iron in the regulation of synthesis of specific proteins in the chloroplast.