MicroRNA-137-3p Protects PC12 Cells Against Oxidative Stress by Downregulation of Calpain-2 and nNOS

The imbalance between excess reactive oxygen species (ROS) generation and insufficient antioxidant defenses contribute to a range of neurodegenerative diseases. High ROS levels damage cellular macromolecules such as DNA, proteins and lipids, leading to neuron vulnerability and eventual death. However, the underlying molecular mechanism of the ROS regulation is not fully elucidated. Recently, an increasing number of studies suggest that microRNAs (miRNAs) emerge as the targets in regulating oxidative stress. We recently reported the neuroprotective effect of miR-137-3p for brachial plexus avulsion-induced motoneuron death. The present study is sought to investigate whether miR-137-3p also could protect PC12 cells against hydrogen peroxide (H₂O₂) induced neurotoxicity. By using cell viability assay, ROS assay, gene and protein expression assay, we found that PC-12 cells exposed to H₂O₂ exhibited decreased cell viability, increased expression levels of calpain-2 and neuronal nitric oxide synthase (nNOS), whereas a decreased miR-137-3p expression. Importantly, restoring the miR-137-3p levels in H₂O₂ exposure robustly inhibited the elevated nNOS, calpain-2 and ROS expression levels, which subsequently improved the cell viability. Furthermore, the suppressive effect of miR-137-3p on the elevated ROS level under oxidative stress was considerably blunted when we mutated the binding site of calpain-2 targted by miR-137-3p, suggesting the critical role of calpain-2 involving the neuroprotective effect of miR-137-3p. Collectively, these findings highlight the neuroprotective role of miR-137-3p through down-regulating calpain and NOS activity, suggesting its potential role for combating oxidative stress insults in the neurodegenerative diseases.

     

番茄种子及其萌发期化感作用分析

采用种子发芽生物测试法,以发芽率、发芽势、发芽指数和活力指数为指标,对番茄(Lycopersicon esculentum Mill.)种子及其萌发期对大白菜[Brassica campestris ssp.pekinensis(Lour.)G.Olsson]、萝卜(Raphanus sativus Linn.)、黄瓜(Cucumis sativus Linn.)和番茄种子萌发的化感效应进行测定,并采用化感效应指数(RI)和化感综合效应(SE)对化感作用进行评价.结果表明:用30 mg·mL-1番茄种子浸提液培养大白菜、萝卜和黄瓜种子,各项发芽指标均低于对照;番茄种子萌发可导致套种的大白菜、萝卜和黄瓜种子的各项发芽指标降低;番茄幼苗分泌物对大白菜、萝卜和黄瓜种子的萌发也均有抑制作用;在4个发芽指标中,对种子活力指数的抑制效应最大.与对照相比,0.5和1.0g番茄幼苗模拟腐解物对大白菜、萝卜和黄瓜种子的发芽率有一定的促进作用,但均导致种子发芽指数和活力指数的降低;而1.5g番茄幼苗模拟腐解物对3种蔬菜种子各项发芽指标的抑制作用最大;随模拟腐解物质量的增加,番茄种子的发芽指标值也逐渐降低.8、16和24 mg·mL-1番茄幼苗浸提液对大白菜、萝卜、黄瓜和番茄种子的萌发有明显抑制作用,随浸提液浓度提高,受试蔬菜种子的各项发芽指标均逐渐降低.总体上看,在番茄种子萌发过程中对受试蔬菜种子萌发的RI值多数为负值、SE值均为负值.研究结果证实:番茄种子含有化感物质,在其萌发过程中分泌与释放化感物质并抑制胚根生长,且除抑制其他植物种子的萌发外还具有自毒作用.     

Highly potent 3-pyrroline mechanism-based inhibitors of bovine plasma amine oxidase and mass spectrometric confirmation of cofactor derivatization

Despite the quinone-dependent copper amine oxidases being described as having the ability to metabolize unbranched primary amines to the corresponding aldehydes, we previously showed that the secondary amines 3-pyrrolines are metabolized as mechanism-based inactivators of bovine plasma amine oxidase (BPAO), and that the 3-(3-nitro-4-methoxyphenyl)-substituted analog was a particularly potent and efficient inactivator. We now show that additional 3-aryl-3-pyrrolines containing highly electron-withdrawing aryl groups (pyridyl, quinolyl, isoquinolyl, and pentafluorophenyl) are some of the most potent inactivators of BPAO reported to date. We also provide mass spectroscopic confirmation of the proposed mechanism of inhibition involving pyrrolylation of the active-site cofactor, through identification by MALDI-TOF and LC-ESI-MS/MS of the (3-arylpyrrol-1-yl)resorcinol derivatives of the cofactor-containing thermolytic peptides.     

A comprehensive review on two-stage integrative schemes for the valorization of dark fermentative effluents

This review provides the alternative routes towards the valorization of dark H₂ fermentation effluents that are mainly rich in volatile fatty acids such as acetate and butyrate. Various enhancement and alternative routes such as photo fermentation, anaerobic digestion, utilization of microbial electrochemical systems, and algal system towards the generation of bioenergy and electricity and also for efficient organic matter utilization are highlighted. What is more, various integration schemes and two-stage fermentation for the possible scale up are reviewed. Moreover, recent progress for enhanced performance towards waste stabilization and overall utilization of useful and higher COD present in the organic source into value-added products are extensively discussed.     

Tpd3‐Pph21 phosphatase plays a direct role in Sep7 dephosphorylation in Candida albicans

Septins are a component of the cytoskeleton and play important roles in diverse cellular processes including cell cycle control, cytokinesis and polarized growth. In fungi, septin organization, dynamics and function are regulated by phosphorylation, and several kinases responsible for the phosphorylation of several septins have been identified. However, little is known about the phosphatases that dephosphorylate septins. Here, we report the characterization of Tpd3, a structural subunit of the PP2A family of phosphatases, in the pathogenic fungus Candida albicans. We found that tpd3Δ/Δ cells are defective in hyphal growth and grow as pseudohyphae under yeast growth conditions with aberrant septin organization. Western blotting detected hyperphosphorylation of the septin Sep7 in cells lacking Tpd3. Tpd3 and Sep7 colocalize at the bud neck and can coimmunoprecipitate. Furthermore, we discovered similar defects in cells lacking Pph21, a catalytic subunit of the PP2A family, and its physical association with Tpd3. Importantly, purified Tpd3‐Pph21 complexes can dephosphorylate Sep7 in vitro. Together, our findings strongly support the idea that the Tpd3‐Pph21 complex dephosphorylates Sep7 and regulates morphogenesis and cytokinesis. The tpd3Δ/Δ mutant is greatly reduced in virulence in mice, providing a potential antifungal target.     

DNA自动测序技术进展

Ｄ Ｎ Ａ 测序是遗传工程的重要技术之一, Ｄ Ｎ Ａ 测序技术的自动化对遗传工程的研究具有重要意义。七十年代末期, Ｓａｎｇｅｒ 和 Ｍａｘａｍ 、 Ｇｉｌｂｅｒｔ分别提出了切实可行的 Ｄ Ｎ Ａ序列测定方法。近二十年来, Ｄ Ｎ Ａ 测序技术发展很快。人们从不同方面对该技术进行了改进,并将许多先进的光学探测方法应用于 Ｄ Ｎ Ａ 测序技术中。目前已出现了许多商品化的 Ｄ Ｎ Ａ 测序仪。     

NaHS对NaHCO3胁迫下黑籽南瓜种子萌发及生理特性的影响

以黑籽南瓜(Cucurbita ficifolia)种子为试材, 研究了外施不同浓度的NaHS对NaHCO₃胁迫下种子萌发及生理特性的影响。结果表明, NaHCO₃胁迫显著抑制了黑籽南瓜种子的发芽率、胚轴长和胚根长, 降低了种子萌发过程中的可溶性糖含量, 抑制了α-淀粉酶、β-淀粉酶、SOD及POD活性。而外施不同浓度的NaHS显著促进了NaHCO₃胁迫下黑籽南瓜萌发种子胚轴和胚根的生长, 提高了可溶性糖含量及α-淀粉酶、β-淀粉酶、SOD和POD活性, 降低了MDA含量; 外施其它盐类(Na₂S、Na₂SO4、NaHSO₄和NaHSO₃)及不同pH值(pH5.8–7.8)的Na₂HPO₄-NaH₂PO₄缓冲液对NaHCO₃胁迫下黑籽南瓜种子的萌发则无影响。外施NaHS可有效缓解NaHCO₃胁迫对黑籽南瓜种子萌发的抑制作用, 其缓解效应可能与其释放的H₂S有关。     

The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its Nα Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

Proteasomes are energy-dependent proteolytic machines. We elaborate here on the previously observed N^α acetylation of the initiator methionine of the α1 protein of 20S core particles (CPs) of Haloferax volcanii proteasomes. Quantitative mass spectrometry revealed this was the dominant N-terminal form of α1 in H. volcanii cells. To further examine this, α1 proteins with substitutions in the N-terminal penultimate residue as well as deletion of the CP “gate” formed by the α1 N terminus were examined for their N^α acetylation. Both the “gate” deletion and Q2A substitution completely altered the N^α-acetylation pattern of α1, with the deletion rendering α1 unavailable for N^α acetylation and the Q2A modification apparently enhancing cleavage of α1 by methionine aminopeptidase (MAP), resulting in acetylation of the N-terminal alanine. Cells expressing these two α1 variants were less tolerant of hypoosmotic stress than the wild type and produced CPs with enhanced peptidase activity. Although α1 proteins with Q2D, Q2P, and Q2T substitutions were N^α acetylated in CPs similar to the wild type, cells expressing these variants accumulated unusually high levels of α1 as rings in N^α-acetylated, unmodified, and/or MAP-cleaved forms. More detailed examination of this group revealed that while CP peptidase activity was not impaired, cells expressing these α1 variants displayed higher growth rates and were more tolerant of hypoosmotic and high-temperature stress than the wild type. Overall, these results suggest that N^α acetylation of α1 is important in CP assembly and activity, high levels of α1 rings enhance cell proliferation and stress tolerance, and unregulated opening of the CP “gate” impairs the ability of cells to overcome salt stress.Proteolysis is important in regulation and protein quality control. Energy-dependent proteases are crucial to early stages of these proteolytic events and include proteasomes, multicatalytic proteases present in all eukaryotes and archaea and in some bacteria. The catalytic component of proteasomes, the 20S core particle (CP), consists of four heptameric rings of α- and β-type subunits stacked as a barrel in an α7β7β7α7 configuration and is essential for growth of archaeal and eukaryotic cells (39, 54). The active sites responsible for peptide bond hydrolysis are formed by N-terminal Thr residues of β-type subunits and are sequestered within the central chamber of the barrel-like structure. Energy-dependent triple-A ATPases, including regulatory particle triple-A ATPases (Rpt) in eukaryotes and proteasome-activating nucleotidases (PAN) in archaea, mediate the unfolding and translocation of substrate proteins through the α-rings for degradation within the CP (39, 40).One major difference between eukaryotic and prokaryotic proteasomal CPs is in the crystal structure of the channel opening formed by the α-rings. Due to partial disorder of the α-subunit N termini, the site of substrate entry appears open at the ends of the cylinders of archaeal and bacterial CPs (e.g., CPs of Thermoplasma acidophilum, Archaeoglobus fulgidus, and Mycobacterium tuberculosis) (13, 15, 27). In contrast, X-ray structures of the CPs of yeast (14) and bovine (45) do not contain this opening. Instead the extreme N termini of the α2, α3, and α4 subunits and the loop structure of α5 fill the central pore in a gate-like structure.Evidence suggests that all CPs are gated, and the major differences observed in the state of the α-ring gate in crystal structures are not physiological. For example, the N-terminal 11 amino acids of the A. fulgidus α subunit, which are not defined by electron density in the CP structure, are more ordered in the 16S “half” proteasome precursor (13). Furthermore, cryoelectron microscopy of the M. tuberculosis CP reveals closed ends that are dependent on the first eight residues of the α-subunit and which diminish peptidolytic activity. Consistent with this, deletion of the N-terminal α-helix (Δ2-12) of the T. acidophilum CP α-subunit abolishes the need for an ATPase (i.e., PAN) in the proteasome-mediated degradation of acid-denatured green fluorescent protein-SsrA or casein (4). In addition, the conserved YDR motif thought to be important in the sterics of α-ring gating is present in all archaeal α-type subunits to date (13). Thus, prokaryotes are thought to gate the α-ring aperture of their proteasomes; however, the physiological consequences of unregulated opening of this gate have not been examined.A gated CP channel formed by the N termini of α-rings may be a general mechanism for regulating the activity of proteasomes. The rate-limiting step in proteasome-mediated protein degradation is translocation of substrates through the α-rings to the active sites contained within the β-rings of the CP (24). Gating is supported by the finding that eukaryotic CPs have no peptidolytic activity in the absence of Rpt proteins or mild chaotropic agents such as sodium dodecyl sulfate (SDS) or heat treatment (9). Furthermore, peptidase activity of the yeast CP is blocked by the N-terminal regions of the α3 subunit. Deletion (Δ2-9) or single substitution (D9A) of N-terminal residues of α3 derepresses this peptidase activity (12).An additional gating mechanism could be employed by posttranslational modifications of the N termini of the α-type subunits. The α-type subunits of CPs are modified by N^α acetylation in several eukaryotes and haloarchaea, including Haloferax volcanii (10, 16, 20, 21, 44). In yeast, N-acetyltransferase 1 (NAT1), the catalytic component of NatA, is responsible for the N^α acetylation of five of the α-type subunits (α1, α2, α3, α4, and α7). Proteasomes purified from a nat1 mutant have twofold-higher chymotrypsin-like peptidase activity in the absence of SDS compared to the wild type, suggesting that N^α acetylation enhances closure of the α-gate (21). In H. volcanii, both α1 and α2 are N^α acetylated on their initiator methionine residue with a subset of α1 not acetylated and instead cleaved by an apparent methionine aminopeptidase (16). A large-scale proteomic survey reveals N^α acetylation is common to other proteasomal α-type proteins of the haloarchaea (10). In this previous survey, the ratios of N^α-acetylated and cleaved forms of the α-type proteins were quantified by spectral counting and estimated to be around 3:1 and 4:3 for Halobacterium salinarum and Natronomonas pharaonis, respectively (10). So far, this existence of these two unique forms of α subunit N termini in the cell simultaneously (initiator methionine N^α acetylated and methionine aminopeptidase [MAP] cleaved) has only been observed in the haloarchaea.In the present study, quantitative tandem mass spectrometry (MS/MS) was used to precisely determine the ratio of the N^α-acetylated to MAP-cleaved forms of the proteasomal α1 protein in H. volcanii. In addition, site-directed mutagenesis was used to examine how the N-terminal penultimate (second) residue and N-terminal α-helix of α1 influence its N^α-acetylated state, CP activity, and cell physiology. Alterations that either fully abolished N^α acetylation or enhanced MAP cleavage of α1 (i) resulted in an increase in CP peptidase activity and (ii) rendered the cells more sensitive to hypoosmotic stress than wild type. In contrast, site-directed changes that generated a mixed population of α1 proteins in various N^α-acetylated states, yet similar N^α-acetylation profiles in CPs to wild type, had profound consequences, including (i) a substantial increase in the levels of α1 protein as heptameric rings, (ii) higher growth rate and cell yield, and (iii) enhanced tolerance of cells to thermal and hypoosmotic stress.