Translation of poly(U) on ribosomes from Streptomyces aureofaciens

Slowly cooled cells of Streptomyces aureofaciens contained mainly tight-couple ribosomes. Maximum rate of polyphenylalanine synthesis on ribosomes of S. aureofaciens was observed at 40°C, while cultures grew optimally at 28°C. Ribosomes of S. aureofaciens differed from those of E. coli in the amount of poly(U) required for maximum synthetic activity. The polyphenylalanine-synthesizing activity of E. coli ribosomes was about 3-times higher than that of S. aureofaciens ribosomes. The addition of protein S1 of E. coli or the homologous protein from S. aureofaciens had no stimulatory effect on the translation of poly(U). In order to localize alteration(s) of S. aureofaciens ribosomes in the elongation step of polypeptide synthesis we developed an in vitro system derived from purified elongation factors and ribosomal subunits. The enzymatic binding of Phe-tRNA to ribosomes of S. aureofaciens was significantly lower than the binding to ribosomes of E. coli. This alteration was mainly connected with the function of S. aureofaciens 50 S subunits. These subunits were not deficient in their ability to associate with 30 S subunits or with protein SL5 which is homologous to L7/L12 of E. coli.     

Lipid II-independent trans Editing of Mischarged tRNAs by the Penicillin Resistance Factor MurM

Streptococcus pneumoniae is a causative agent of nosocomial infections such as pneumonia, meningitis, and septicemia. Penicillin resistance in S. pneumoniae depends in part upon MurM, an aminoacyl-tRNA ligase that attaches l-serine or l-alanine to the stem peptide lysine of Lipid II in cell wall peptidoglycan. To investigate the exact substrates the translation machinery provides MurM, quality control by alanyl-tRNA synthetase (AlaRS) was investigated. AlaRS mischarged serine and glycine to tRNA^Ala, as observed in other bacteria, and also transferred alanine, serine, and glycine to tRNA^Phe. S. pneumoniae tRNA^Phe has an unusual U4:C69 mismatch in its acceptor stem that prevents editing by phenylalanyl-tRNA synthetase (PheRS), leading to the accumulation of misaminoacylated tRNAs that could serve as substrates for translation or for MurM. Although the peptidoglycan layer of S. pneumoniae tolerates a combination of both branched and linear muropeptides, deletion of MurM results in a reversion to penicillin sensitivity in strains that were previously resistant. However, because MurM is not required for cell viability, the reason for its functional conservation across all strains of S. pneumoniae has remained elusive. We now show that MurM can directly function in translation quality control by acting as a broad specificity lipid-independent trans editing factor that deacylates tRNA. This activity of MurM does not require the presence of its second substrate, Lipid II, and can functionally substitute for the activity of widely conserved editing domain homologues of AlaRS, termed AlaXPs proteins, which are themselves absent from S. pneumoniae.     

水稻种子发育过程中Poly(A)RNA和蛋白质水平的变化

我们用[~3H]—Poly(U)饱和杂交的方法分析了水稻种子发育过程中Poly(A)含量和Poly(A)RNA水平的变化。胚乳发育过程中,Poly(A)含量和Poly(A)RNA水平均于开花后11天达到高峰,比蛋白质高峰出现时间约早10天。随着胚乳的成熟,蛋白质水平在开花后6～21天持续增长。但 Poly(A)含量和Poly(A)RNA水平却急剧下降。因此,在胚乳发育早期合成的Poly(A)RNA中,可能有部分不是直接用于蛋白质的合成。在胚的发育过程中,Poly(A)含量和Poly(A)RNA水平分别出现三次高峰。开花后30天,每胚含有5.94ng Poly(A)RNA,约占胚总RNA的0.097％,为稻胚中贮存的mRNA存在提供了一个直接的证据。     

Poly(etheno ADP-ribose) blocks poly(ADP-ribose) glycohydrolase activity

Poly(ADP-ribose) is a biopolymer synthesized by poly(ADP-ribose) polymerases. Recent findings suggest the possibility for modulation of cellular functions including cell death and mitosis by poly(ADP-ribose). Derivatization of poly(ADP-ribose) may be useful for investigating the effects of poly(ADP-ribose) on various cellular processes. We prepared poly(etheno ADP-ribose) (poly(epsilonADP-ribose)) by converting the adenine moiety of poly(ADP-ribose) to 1-N(6)-etheno adenine residues. Poly(epsilonADP-ribose) is shown to be highly resistant to digestion by poly(ADP-ribose) glycohydrolase (Parg). On the other hand, poly(epsilonADP-ribose) could be readily digested by phosphodiesterase. Furthermore, poly(epsilonADP-ribose) inhibited Parg activity to hydrolyse ribose-ribose bonds of poly(ADP-ribose). This study suggests the possibility that poly(epsilonADP-ribose) might be a useful tool for studying the poly(ADP-ribose) dynamics and function of Parg. This study also implies that modification of the adenine moiety of poly(ADP-ribose) abrogates the susceptibility to digestion by Parg.     

A kinetic analysis of cyanine selectivity: CCyan2 and Cyan40 intercalation into poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC)

A T-jump investigation of the binding of Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC) is performed at I = 0.1M (NaCl), 25 degrees C and pH 7. Two kinetic effects are observed for both systems. The binding process is discussed in terms of the sequence D + P <==> P,D <==> PD(I) <==> PD(II), which leads first to fast formation of a precursor complex P,D and then to a partially intercalated complex PD(I) which converts to the fully intercalate complex PD(II). Concerning CCyan2 the rate parameters depend on the polymer nature and their analysis shows that in the case of poly(dG-dC) x poly(dG-dC) the most stable bound form is the fully intercalated complex PD(II), whereas in the case of poly(dA-dT) x poly(dA-dT) the partially intercalated complex PD(I) is the most stable species. Concerning Cyan40, the rate parameters remain unchanged on going from A-T to G-C indicating that this dye is unselective.     

Electroosmotic properties of microfluidic channels composed of poly(dimethylsiloxane)

Microfluidic devices fabricated from polymers exhibit great potential in biological analyses. Poly(dimethylsiloxane) (PDMS) has shown promise as a substrate for rapid prototyping of devices. Despite this, disagreement exists in the literature as to the ability of PDMS to support electroosmotic (EO) flow and the stability of that flow over time. We demonstrate that in low ionic strength solutions near neutral in pH, oxidized PDMS had a four-fold greater EO mobility (μ_eo) compared to native PDMS. The greater μ_eo was maintained irrespective of whether glass or PDMS was used as a support forming one side of the channel. This enhanced μ_eo was preserved as long as the channels were filled with an aqueous solution. Upon exposure of the channels to air, the mobility decreased by a factor of two with a half-life of 9 h. The EO properties of the air-exposed, oxidized PDMS were regenerated by exposure to strong base. High ionic strength, neutral in pH buffers compatible with living eukaryotic cells diminished the EO flow in the oxidized PDMS devices to a much greater extent than in the native PDMS devices. For analyses utilizing intact and living cells, oxidation of PDMS may not be an effective strategy to substantially increase the μ_eo.     

Altered poly(ADP-ribose) metabolism impairs cellular responses to genotoxic stress in a hypomorphic mutant of poly(ADP-ribose) glycohydrolase

Genotoxic stress activates nuclear poly(ADP-ribose) (PAR) metabolism leading to PAR synthesis catalyzed by DNA damage activated poly(ADP-ribose) polymerases (PARPs) and rapid PAR turnover by action of nuclear poly(ADP-ribose) glycohydrolase (PARG). The involvement of PARP-1 and PARP-2 in responses to DNA damage has been well studied but the involvement of nuclear PARG is less well understood. To gain insights into the function of nuclear PARG in DNA damage responses, we have quantitatively studied PAR metabolism in cells derived from a hypomorphic mutant mouse model in which exons 2 and 3 of the PARG gene have been deleted (PARG-Delta2,3 cells), resulting in a nuclear PARG containing a catalytic domain but lacking the N-terminal region (A domain) of the protein. Following DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we found that the activity of both PARG and PARPs in intact cells is increased in PARG-Delta2,3 cells. The increased PARG activity leads to decreased PARP-1 automodification with resulting increased PARP activity. The degree of PARG activation is greater than PARP, resulting in decreased PAR accumulation. Following MNNG treatment, PARG-Delta2,3 cells show reduced formation of XRCC1 foci, delayed H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. Our results show that a precise coordination of PARPs and PARG activities is important for normal cellular responses to DNA damage and that this coordination is defective in the absence of the PARG A domain.     

Role of poly(ADP-ribose) glycohydrolase silencing in DNA hypomethylation induced by benzo(a)pyrene

Benzo(a)pyrene (BaP) is a known carcinogen cytotoxic which can trigger extensive cellular responses. Many evidences suggest that inhibitors of poly(ADP-ribose) glycohydrolase (PARG) are potent anticancer drug candidates. However, the role of PARG in BaP carcinogenesis is less understood. Here we used PARG-deficient human bronchial epithelial cell line (shPARG cell) as an in vitro model, and investigated the role of PARG silencing in DNA methylation pattern changed by BaP. Our study shows, BaP treatment decreased global DNA methylation levels in 16HBE cells in a dose-dependent manner, but no dramatic changes were observed in shPARG cells. Further investigation revealed PARG silencing protected DNA methyltransferases (DNMTs) activity from change by BaP exposure. Interestingly, Dnmt1 is PARylated in PARG-null cells after BaP exposure. The results show a role for PARG silencing in DNA hypomethylation induced by BaP that may provide new clue for cancer therapy.     

Misregulation of poly(ADP-ribose) polymerase-1 activity and cell type-specific loss of poly(ADP-ribose) synthesis in the cerebellum of aged rats

Protein modification by ADP-ribose polymers is a common regulatory mechanism in eukaryotic cells and is involved in several aspects of brain physiology and physiopathology, including neurotransmission, memory formation, neurotoxicity, ageing and age-associated diseases. Here we show age-related misregulation of poly(ADP-ribose) synthesis in rat cerebellum as revealed by: (i) reduced poly(ADP-ribose) polymerase-1 (PARP-1) activation in response to enzymatic DNA cleavage, (ii) altered protein poly(ADP-ribosyl)ation profiles in isolated nuclei, and (iii) cell type-specific loss of poly(ADP-ribosyl)ation capacity in granule cell layer and Purkinje cells in vivo. In particular, although PARP-1 could be detected in virtually all granule cells, only a fraction of them appeared to be actively engaged in poly(ADP-ribose) synthesis and this fraction was reduced in old rat cerebellum. NAD(+), quantified in tissue homogenates, was essentially the same in the cerebellum of young and old rats suggesting that in vivo factors other than PARP-1 content and/or NAD(+) levels may be responsible for the age-associated lowering of poly(ADP-ribose) synthesis. Moreover, PARP-1 expression was substantially down-regulated in Purkinje cells of senescent rats.     

聚乳酸材料在不同土壤环境中生物降解的菌群结构分析

【目的】评价聚乳酸(Polylactic acid,PLA)材料在不同土壤环境中自然降解的效果,通过对3种不同土壤菌群结构的分析,找到能够对聚乳酸材料有降解作用的优势菌群。【方法】通过扫描电镜、断裂拉伸强度和CO2释放量测定来评价3种土壤对PLA材料的降解效果,并运用高通量测序技术,对3种土壤细菌群落进行基因组测序分析,检测3个样本细菌群落的差异性。【结果】PLA材料在沼泽地、芒果林地和稻田中的生物降解率分别为13.7%、10.6%和4.5%。3种土壤的样品分别获得11 110、11 236和8 848个OTU,共涉及细菌域的9个主要门和16个主要科。其中沼泽地土壤的微生物群落丰富度和多样性最高,稻田土壤最低。【结论】结合土壤的降解效果,土壤中生物群落丰富度和多样性越高,对PLA材料的降解作用越好。同时变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)是降解聚乳酸材料的优势菌群。在科水平上,黄杆菌科(Flavobacteriaceae)、丛毛单胞菌科(Comamonadaceae)和噬纤维菌科(Cytophagaceae)的微生物对聚乳酸材料的降解最有潜力。这一研究成果为能有效降解聚乳酸材料的微生物资源的开发提供了理论依据。     

Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V_max) and the michaelis constant (k_m) of PARG reaction were 4.46 μM and 128.33 μmol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 μM. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.     

Ecdysone receptor-dependent gene regulation mediates histone poly(ADP-ribosyl)ation

While the ecdysone dependency of puff formation in giant polytene chromosomes from fly salivary glands has been well documented, the molecular mechanisms underlying this process remain unknown. However, it does appear to involve chromatin remodeling and modification mediated by ecdysone receptor (EcR). As Drosophila poly(ADP-ribose) polymerase (dPARP) has recently been reported to be involved in ecdysone-induced puff formation, we decided to test the possible role of dPARP in ligand-induced dEcR transactivation in an insect system. dPARP co-activated the ligand-induced transactivation function of EcR in the insect cell line S2, and appeared to physically interact with EcR in a ligand-dependent manner. ChIP analysis of an EcR target gene promoter revealed ligand-dependent recruitment of dPARP with poly(ADP-ribosyl)ation of histones in the EcR binding site and, surprisingly, also in a distal region of the promoter. Our results indicated that EcR-mediated gene regulation may be coupled with chromatin modification through poly(ADP-ribosyl)ation.     

Haploinsufficiency of poly(ADP-ribose) polymerase-1-mediated poly(ADP-ribosyl)ation for centrosome duplication

The centrosome plays a vital role in maintaining chromosomal stability. Known as the microtubule organizing center, the centrosome is involved in the formation of spindle poles during mitosis, which ensures the distribution of the correct number of chromosomes to daughter cells. Aberrant centrosome duplication could cause centrosome amplification and chromosomal instability. We have previously shown that poly(ADP-ribose) polymerase-1 (PARP-1) is important for centrosome function and chromosomal stability. In this study, we used PARP-1(+/+), PARP-1(+/-) and PARP-1(-/-) primary mouse embryonic fibroblasts and found that the level of PARP-1 gene dosage correlates with PARP activity and the in vivo level of poly(ADP-ribosyl)ation, which could explain the mechanism by which PARP-1 haploinsufficiency affects centrosome duplication and chromosomal stability. Our results emphasize that correct regulation of poly(ADP-ribosyl)ation levels in vivo is important for maintenance of proper centrosome duplication and chromosomal stability.     

Acid-tolerant poly(3-hydroxybutyrate) hydrolases from moulds

Abstract We have isolated some mould strains that can grow under acid conditions with poly(3-hydroxybutyrate) (PHB) as sole carbon source, and secrete PHB hydrolases active at pH values at least down to 3. An improved assay method for such enzymes using a pH stat has been developed, and used to determine the dependence of reaction rate on enzyme and polymer concentrations. The implications of these kinetic properties of the PHB hydrolase for its mode of action are discussed.     

Regulation of poly(A) polymerase activity and poly(A)+ RNA in germinated wheat embryos under conditions of pathogenesis

The induction of poly(A) polymerase was accompanied by a rise in the level of poly(A)⁺ RNA during early germination of excised wheat embryos (48 h). Fractionation of this RNA-processing enzyme by acrylamide gel electrophoresis and also by molecular sieving on Sephadex G-200 revealed a single molecular form of poly(A) polymerase with a molecular weight of 125 000. Wheat poly(A) polymerase specifically catalyzed the incorporation of [³H]AMP from [³H]ATP into the polyadenylate product only in the presence of primer RNA. Substitution of [³H]ATP by other labelled nucleoside triphosphates, such as [³H]GTP, [³H]UTP or [α-³²P]CTP in the assay mixture did not yield any labelled polynucleotide reaction product. The ³H-labelled reaction product was retained on poly(U)-cellulose affinity column and was not degraded by RNAase A and RNAase T₁ treatment. In addition, the nearest-neighbour frequency analysis of the ³²P-labelled reaction product predominantly yielded [³²P]AMP. Thus, characterization of the reaction product clearly indicated its polyadenylate nature. The average chain length of the [³H]poly(A) product was 26 nucleotides. Infection of germinating wheat embryos by a fungal pathogen (Drechslera sorokiana) brought about a severe inhibition (62–79%) of poly(A) polymerase activity. Concurrently, there was a parallel decrease (73%) in the level of poly(A)⁺ RNA. Inhibition of poly(A) polymerase activity in infected embryos could be due to enzyme inactivation, which in turn brought about a downward shift in the level of poly(A)⁺ RNA. The crude extract of the cultured pathogen contains a non-dialysable, heat-labile factor, which, along with a ligand, inactivates (65–74%) poly(A) polymerase in vitro. The fungal extracts also contained a dialysable, heat-stable stimulatory effector which activated wheat poly(A) polymerase (3.6–4.0-fold stimulation) in vitro. However, the stimulatory fungal effector was not expressed in vivo, but was detectable after the inhibitory fungal factor had been destroyed by heat-treatment in our in vitro experiments.