Regulatory perspective of antibiotic biosynthesis in Streptomyces

<正>Streptomycetes are Gram-positive bacteria with high GC DNA content. They produce the most abundant secondary metabolites including over two-thirds of the clinically used antibiotics of natural origin (Barka et al., 2016), for example,the important broad-spectrum antimicrobials oxytetracycline(OTC) and chlortetracycline, which are the tetracycline antibiotics, produced by Streptomyces rimosus and Strepto-     

皱纹盘鲍的染色体研究

本文报道皱纹盘鲍的染色体制备方法及核型分析结果。皱纹盘鲍的2n=36,可配为18对,中央着丝点(M)的11对,即1,3,6,7,9,11,12,13,14,16,17号染色体。亚中央着丝粒(Sm)的7对,即2,4,5 8,15,18号染色体,NF=72。染色体的长度呈连续递变。其中相对长度最长的1号染色体为7.09,最短的18号染色体4.11,单倍体总长687.05。     

Determination of lactate dehydrogenase in human erythrocytes by capillary electrophoresis with electrochemical detection

Capillary electrophoresis (CE) was employed to analyze lactate dehydrogenase (LDH) in human erythrocytes using an amperometric detector with a carbon fiber micro-disk bundle electrode. LDH activity was measured by determining the amount of NADH generated by LDH through a enzyme-catalyzed reaction between NAD(+) and lithium lactate. The factors influencing the enzyme-catalyzed reaction, separation and detection were examined and optimized. The following conditions were suitable for the determination of LDH: running buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 7.5); separation voltage, 20.0 kV; detection potential, 1.00 V (versus saturated calomel electrode (SCE)). The conditions of enzyme-catalyzed reaction were: reaction buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 9.3); substrates, 5.0 x 10(-2)mol/l lithium lactate and 5.0 x 10(-3)mol/l NAD(+); reaction time, 10 min. The concentration limit of detection (LOD) of the method was 0.017 U/ml at a signal-to-noise (S/N) ratio of 3, which corresponded to 1.10 x 10(-10)mol/l, and the mass LOD was 2 x 10(-20)mol. The linear dynamic range was 0.039-4.65 U/ml for the injection voltage of 5.0 kV and injection time of 10s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the electrophoretic peak area. The method was applied to determine LDH in human erythrocytes. The recovery of the method was between 98 and 101%.     

Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction

MDM2 is an important negative regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-molecule inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.     

Transferrin-Directed Internalization and Cycling of Transferrin Receptor 2

Transferrin receptor 2 (TfR2) is a homologue of transferrin receptor 1 (TfR1) but has distinct functions from TfR1 in iron homeostasis. In keeping with its proposed role in iron sensing, previous studies showed that TfR2 has a short half-life and that holo-Tf stabilizes TfR2 by redirecting it from a degradative pathway to a recycling pathway. In this study, we characterized how the endocytosis, recycling and degradation of TfR2 relates to its function and differs from TfR1. TfR2 endocytosis was adaptor protein-2 (AP-2) dependent. Flow cytometry analysis showed that TfR1 and TfR2 utilized the same endocytic pathway only in the presence of holo-Tf, indicating that holo-Tf alters the interaction of TfR2 with the endocytic machinery. Unlike TfR1, phosphofurin acidic cluster sorting protein 1 (PACS-1) binds to the cytoplasmic domain of TfR2 and data suggest that PACS-1 is involved in the TfR2 recycling. Depletion of TSG101 by siRNA or expression of a dominant negative Vps4 inhibited TfR2 degradation, indicating that TfR2 degradation occurs through a multivesicular body (MVB) pathway. TfR2 degradation is not mediated through ubiquitination on the single lysine (K31) in the cytoplasmic domain or on the amino terminal residue. No ubiquitination of TfR2 by HA-ubiquitin was detected, indicating a lack of direct TfR2 ubiquitination involvement in its degradation.     

PML/RARalpha fusion protein mediates the unique sensitivity to arsenic cytotoxicity in acute promyelocytic leukemia cells: Mechanisms involve the impairment of cAMP signaling and the aberrant regulation of NADPH oxidase

Acute promyelocytic leukemia (APL) cells are characterized by PML/RARalpha fusion protein, high responsiveness to arsenic trioxide (ATO)-induced cytotoxicity and an abundant generation of reactive oxygen species (ROS). In this study we investigated the association among these three features in APL-derived NB4 cells. We found that NADPH oxidase-derived ROS generation was more abundant in NB4 cells compared with monocytic leukemia U937 cells. By using PR9, a sub-line of U937 stably transduced with the inducible PML/RARalpha expression vectors, we attributed disparities on ROS generation and ATO sensitivity to the occurrence of PML/RARalpha fusion protein, since PML/RARalpha-expressing cells appeared higher NADPH oxidase activity, higher ROS level and higher sensitivity to ATO. On the other hand, the basal intensity of cAMP signaling pathway was compared between NB4 and U937 as well as between PR9 cells with or without PML/RARalpha, demonstrating that PML/RARalpha-expressing cells had an impaired cAMP signaling pathway which relieved its inhibitory effect on NADPH oxidase derived ROS generation. In summary, the present study demonstrated the correlation of PML/RARalpha with cAMP signaling pathway, NADPH oxidase and ROS generation in APL cells. PML/RARalpha that bestows NB4 cells various pathological features, paradoxically also endows these cells with the basis for susceptibility to ATO-induced cytotoxcity.     

Purification and properties of alkaline phosphatase of silkworm Bombyx mori

Alkaline phosphatase(AKP),from the succus entericus of silkworm,was purified using 10%-50% ammonium sulfate fractions,ion exchange chromatography Of DEAE-Sepharose,and size exclusion chromatography of Sephacryl S-200.The purification fold was 464 times and specified activity was 3936 U/mg.Optimum pH value of the phosphatase was 10.5,and was stable between pH 7.5 and 11.The optimum temperature of the phosphatase was 40℃ and it was unstable over 50℃.Km value of the phosphatase was 1.25 mmol/L.In a given condition,the phosphatase was selectively modified by PCMB,NBS,PMSE TNBS,SUAN,DTT,BrAc,and IAc,the results indicate that PMSF,SUA,BrAc,IAc,and TNBS could Obviously inhibit the activity of the phosphatase,and the degree of inhibition depended on the concentration of these reagents.There was little effect on the activity of phosphatase after treatment by PMSF,DTT,and NBT.We primarily conclude that mercapto and imidazole are essential for AKP from silkworm.Also,Lys residue and disulfide bands are necessary to protect the catalysis of the AKP.     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A₂ (PLA₂) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C₁₈ chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A₂s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A₂s are distinct from the other subgroups (D49 PLA₂, S49 PLA₂ and K49 PLA₂) and represent a unique subgroup of snake venom group II PLA₂, named N49 PLA₂ subgroup.     

资源限制与发展停滞：传统社会的生态学分析

资源限制与发展停滞：传统社会的生态学分析王建革（复旦大学历史地理研究所,上海２００４３３）ＲｅｓｏｕｒｃｅｓＬｉｍｉｔａｔｉｏｎａｎｄＳｌｏｗＤｅｖｅｌｏｐｍｅｎｔ：ＡｎＥｃｏｌｏｇｉｃａｌＡｎａｌｙｓｉｓｏｆＴｒａｄｉｔｉｏｎａｌＳｏｃｉｅｔｙ．Ｗ...