转录因子TCF7L2在HePG2细胞IDE表达调控中的作用

目的 TCF7L2是一种重要的转录因子,与2型糖尿病(T2DM)发生发展密切相关.本研究探讨慢病毒介导的RNA干扰抑制TCF7L2基因表达对人肝癌细胞株HePG2胰岛素降解酶(IDE)基因的影响.方法 以人TCF7L2 mRNA编码序列作为干扰靶点,构建TCF7L2特异性短发卡RNA慢病毒表达载体(LV-TCF7 L2-shRNA)感染HePG2细胞.应用实时定量PCR及Western blot检测转染后TCF7L2与IDE表达的变化.结果 成功构建TCF7L2 shRNA慢病毒载体LV-TCF7L2-shRNA.qPCR及Westem blot结果显示干扰组HePG2细胞TCF7L2和IDE mRNA及蛋白的表达水平较空白组及阴性对照组显著降低(P＜0.05).结论 LV-TCF7L2-shRNA载体有效地抑制了IDE的表达,结果证明TCF7L2是IDE表达调控中重要的转录因子,为探讨TCF7L2与IDE在2型糖尿病发病机制中的作用奠定了基础.     

The E-site story: the importance of maintaining two tRNAs on the ribosome during protein synthesis

In the sixties James Watson suggested a twosite model for the ribosome comprising the P site for the peptidyl transfer RNA (tRNA) before peptide-bond formation and the A site, where decoding takes place according to the codon exposed there. In the eighties a third tRNA binding site was detected, the E site, which was specific for deacylated tRNA and turned out to be a universal feature of ribosomes. However, despite having three tRNA binding sites, only two tRNAs occupy the ribosome at a time during protein synthesis: at the A and P sites before translocation (PRE state) and at the P and E sites after translocation (POST state). The importance of having two tRNAs in the POST state has been revealed during the last 25 years, showing that the E site contributes two fundamental features: (i) the fact that incorporation of a wrong amino acid is not harmful for the cell (only 1 in about 400 misincorporations destroys the function of a protein) stems from the presence of an E-tRNA; (ii) maintenance of the reading frame is one of the most remarkable achievements of the ribosome, essential for faithful translation of the genetic information. The presence of the POST state E-tRNA prevents loss of the reading frame. Received 14 March 2006; received after revision 8 June 2006; accepted 4 August 2006     

L1Tc non-LTR retrotransposons from Trypanosoma cruzi contain a functional viral-like self-cleaving 2A sequence in frame with the active proteins they encode

A comparative analysis of 40 Trypanosoma cruzi L1Tc elements showed that the 2A self-cleaving sequence described in viruses is present in them. Of these elements, 72% maintain the canonical 2A motif (DxExNPGP). A high percentage has a conserved point mutation within the motif that has not been previously described. In vitro and in vivo expression of reporter polyproteins showed that the L1Tc2A sequence is functional. Mutations within certain L1Tc2A sequences affect the efficiency of the cleavage. The data indicate that the L1Tc2A sequence may be influencing the L1Tc enzymatic machinery determining the composition and level of the translated products. The residues located immediately upstream of the 2A consensus sequence increase the cleaving efficiency and appear to stabilize the relative amount of translated products. These authors contributed equally to this work. Received 26 January 2006; received after revision 11 April 2006; accepted 21 April 2006     

K252a: A new blocker of the cell-cycle at G1 phase in a human hepatoma cell line

The administration of 200 nM K252a to HuH7 suppressed the proliferation of the cells almost completely. The uptake of [³H]thymidine was inhibited, and flow cytometry revealed only one peak at 2C on day 3 after treatment with 100 nM K252a. The expression of proto-oncogene c-myc was not reduced. Despite the blockage at G1, both the size of the cells and the amount of cell protein had increased by 4 times by day 3 after treatment with K252a, while the cells secreted albumin and -fetoprotein into the medium as usual. These results show that K252a can increase the cell size of HuH7 without losing its function by blocking the cell cycle at G1 phase.     

Viewing past science from the point of view of present science,thereby illuminating both: Philosophy versus experiment in the work of Robert Boyle

The seventeenth century witnessed the replacement of an Aristotelian worldview by a mechanical one. It also witnessed the beginnings of significant experimental enquiry. Alerted by the fact that the methods involved in the latter, but not in the former, resemble those employed in later science, I argue the historical case that the emergence of the mechanical worldview and the emergence of science were not closely related and that it was the latter that was to develop into science as we have come to know it. The details are explored in the context of the philosophical and experimental work of Robert Boyle and the relationship between them.     

New endo-beta-1,4-glucanases from the parabasalian symbionts,Pseudotrichonympha grassii and Holomastigotoides mirabile of Coptotermes termites

An endo-β-1,4-glucanase (EG) was purified from the hindgut of an Australian mound-building termite, Coptotermes lacteus. The hindgut extract had a peak separate from those for extracts obtained from the salivary glands and the midgut based on sephacryl S-200 gel chromatography, and also demonstrated an origin different from the endogenous EGs of the termite itself. The recovery was further purified by SDS-PAGE, and its N-terminal amino acid sequence analyzed. This showed high homology to EGs from glycoside hydrolase family (GHF) 7. PCR-based cloning methods were applied to the hindgut contents of C. lacteus and individual protozoan symbionts from C. formosanus. cDNAs encoding putative EGs homologous to GHF7 members were then identified. The functionality of one of the putative proteins was confirmed by its expression in Escherichia coli. Received 18 September 2002; accepted 20 September 2002 RID="*" ID="*"Corresponding author.     

Arresting the mitotic oscillator and the control of cell proliferation: insights from a cascade model for cdc2 kinase activation

We consider a minimal cascade model previously proposed¹¹ for the mitotic oscillator driving the embryonic cell division cycle. The model is based on a bicyclic phosphorylation-dephosphorylation cascade involving cyclin and cdc2 kinase. By constructing stability diagrams showing domains of periodic behavior as a function of the maximum rates of the kinases and phosphatases involved in the two cycles of the cascade, we investigate the role of these converter enzymes in the oscillatory mechanism. Oscillations occur when the balance of kinase and phosphatase rates in each cycle is in a range bounded by two critical values. The results suggest ways to arrest the mitotic oscillator by altering the maximum rates of the converter enzymes. These results bear on the control of cell proliferation.     

Luffolide,a novel anti-inflammatory terpene from the spongeLuffariella sp.

Summary Luffolide (4) is a minor metabolite of the spongeLuffariella sp. from Palau. The structure of luffolide was determined by single crystal X-ray analysis. Luffolide is relatively unstable and undergoes a complex cyclization reaction to give the hexacyclic products5 and6. Luffolide (4) has some of the anti-inflammatory properties of manoalide (1): this may help to define the chemical reaction between manoalide (1) and phospholipase A₂.All crystallographic calculations were done on a PRIME 9950 computer operated by the Cornell Chemistry Computing Facility. Principal programs employed were: FOBS, a data reduction program by G.D. Van Duyne, Cornell University, 1987; MULTAN 80, and RANTAN 80, systems of computer programs for the automatic solution of crystal structures from X-ray diffraction data (locally modified to perform all Fourier calculations including Patterson syntheses) written by P. Main, S. E. Hull, L. Lessinger, G. Germain, J. P. Declercq and M. M. Woolfson, University of York, England, 1980 BDLS, an, anisotropic block diagonal least squares refinement written by K. Hirotsu, E. Arnold, and G. D. Van Duyne, Cornell University, 1987; PLUTO 78, a locally modified crystallographic illustration program by W. D. S. Motherwell, Cambridge Crystallographic Data Centre, 1978; and BOND, a program to calculate molecular parameters and prepare tables written by K. Hirotsu and G. Van Duyne, Cornell University, 1985.Acknowledgment. We thank the Government of the Republic of Palau for a scientific research permit. We thank Dr Klaus Rützler, Smithsonian Institution, Washington, D.C. for identifying the sponge and Mary Kay Harper for performing additional bioassays. This research was supported by grants from the Sea Grant College Programs of California [Projects R/MP-30 to DJF) and R/MP-31 (to RSJ)] and New York (to JC) and the National Institutes of Health (CA 24487 to JC).     

Nature of the FeO2 bonding in myoglobin: An overview from physical to clinical biochemistry

Summary The iron(II)-dioxygen bond in myoglobin and hemoglobin is a subject of wide interest. Studieas range from examinations of physical-chemical properties dependent on electronic structure, to investigations of stability as a function of oxygen supply. Stability properties are of particular importance in vivo, since the oxygenated form is known to be oxidized easily to the ferric form, which cannot be oxygenated and is therefore physiologically inactive.Kinetic and thermodynamic studies of the stability of native oxymyoglobin have revealed a new feature in FeO₂ bonding. In vivo. the iron center is always subject to a nucleophilic attack of the water molecule or hydroxyl ion, which can enter the heme pocket from the surrounding solvent, and thereby irreversibly displace the bound dioxygen from MbO₂ in the form of O ₂ ^– so that the iron is converted to the ferric form. A free energy diagram for the potential reactions of FeO₂ visualizes myoglobin as a molecular structure that can provide in solution the delicate balance of kinetic and thermodynamic factors necessary to stabilize reversible oxygenation, as opposed to irreversible autoxidation to metmyoglobin.     

Chemotherapy and immunotherapy of malignant glioma: molecular mechanisms and clinical perspectives

Despite the considerable progress in modern tumor therapy, the prognosis for patients with glioblastoma, the most frequent malignant brain tumor, has not been substantially improved. Although cytoreductive surgery and radiotherapy are the mainstays of treatment for malignant glioma at present, novel cytotoxic drugs and immunotherapeutic approaches hold great promise as effective weapons against these malignancies. Thus, great efforts are being made to enhance antitumoral efficacy by combining various cytotoxic agents, by novel routes of drug administration, or by combining anticancer drugs and immune modulators. Immunotherapeutic approaches include cytotoxic cytokines, targeted antibodies, and vaccination strategies. However, the success of most of these experimental therapies is prevented by the marked molecular resistance of glioma cells to diverse cytotoxic agents or by glioma-associated immunosuppression. One promising experimental strategy to target glioma is the employment of death ligands such as CD95 (Fas/Apo1) ligand or Apo2 ligand (TRAIL). Specific proapoptotic approaches may overcome many of the obvious obstacles to a satisfactory management of malignant brain tumors. Received 8 March 1999; received after revision 27 May 1999; accepted 14 June 1999     

Natriuretic peptide hormones promote radial water movements from the xylem of Tradescantia shoots

Immunological evidence suggests that plants, like vertebrates, contain natriuretic peptides (NPs) and that rat atrial NP (rANP) binds specifically to plant membranes and promotes concentration and conformation-dependent stomatal opening. Stomatal opening and specific increases in cGMP levels were also observed in response to immunoreactive plant NP (irPNP). Here we report that both 1 μM rANP and irPNP (100 ng total protein/100 μL) significantly increase radial water movements out of the xylem of shoots of Tradescantia multiflora. Enhanced radial water movements are also observed in response to the cell permeant cGMP analogue 8-Br-cGMP (100 nM). The water channel inhibitor mercuric chloride (HgCl₂) significantly inhibits radial water movements at concentrations of 50 μM, while the presence of 10 μM 2-hydroxyethylmercaptoethanol (ME) prevents the inhibitory effect of the mercurial. The guanylate cyclase inhibitor LY 83583 at a concentration of 20 μM and sodium azide (NaN₃) at concentrations of ≥ 1 μM both also reduce radial water movements. We therefore conclude that the regulation of radial water movement out of the xylem involves modulation of cGMP levels, water channels and respiration-dependent processes. In addition, we propose that NPs have a critical role to play in radial water movements out of the xylem and speculate that as in vertebrates, NP effects might, at least in part, be mediated via the regulation of guanylate cyclases and water channels. Received 15 June 1998; received after revision 7 August 1998; accepted 26 August 1998     

The membrane component of the ribosomal bodies from the lizardLacerta sicula: a freeze-etching study

Summary We have studied, using the freeze-etching technique, the cytomembranes of the flat cystenae, regularly interposed between the cristalline sheets of ribosomes, inside the ribosomal bodies of the hibernating lizardLacerta sicula. The results show the presence, on the protoplasmic face (PF), of numerous IMPs (intramembrane particles) which can be separated into two populations. The flatter ones appear to be arranged in a regular pattern, with the support of the optical diffraction analysis, can be correlated with the disposition of the ribosomes in the cristalline sheets.6 October 1986     

The Arp2/3 complex: a central regulator of the actin cytoskeleton

In recent years the Arp2/3 complex has emerged as a central regulator of actin dynamics, assembling and cross-linking actin filaments to produce a diverse array of cellular structures. Here I discuss our current state of knowledge about this actin-remodelling machine. The predicted structure of the Arp2/3 complex can be directly correlated with its ability to nucleate, cap and cross-link actin filaments. A growing family of Arp2/3 complex activators such as the WASP family, type I myosins, and the newly identified activators cortactin and Abp1p tightly regulate this activity within the cell. Localised activation of the Arp2/3 complex produces structures such as lamellipodia or actin patches via a process termed dendritic nucleation. Furthermore, several pathogenic microorganisms have evolved strategies to 'hijack' the Arp2/3 complex to their own advantage. Finally, I discuss some of the questions which remain unanswered about this fascinating complex. Received 2 April 2001; received after revision 15 May 2001; accepted 18 May 2001     

Plakorin,a potent Ca2+-ATPase activator from the Okinawan marine spongePlakortis sp.

Summary A new cyclic peroxide, plakorin, which is a potent sarcoplasmic reticulum (SR) Ca^a+-ATPase activator has been isolated from the Okinawan marine spongePlakortis sp., its structure was elucidated on the basis of spectral data.Acknowledgments. We thank Ms M. Hamashima and Ms A. Muroyama for their technical assistance.     

High molecular weight FGF2: the biology of a nuclear growth factor

Fibroblast growth factor 2 (FGF2) is one of the most studied growth factors to date. Most attention has been dedicated to the smallest, 18kDa FGF2 variant that is released by cells and acts through activation of cell-surface FGF-receptor tyrosine kinases. There are, however, several higher molecular weight (HMW) variants of FGF2 that rarely leave their producing cells, are retained in the nucleus and act independently of FGF-receptors (FGFR). Despite significant evidence documenting the expression and intracellular trafficking of HMW FGF2, many important questions remain about the physiological roles and mechanisms of action of HMW FGF2. In this review, we summarize the current knowledge about the biology of HMW FGF2, its role in disease and areas for future investigation. Received 28 July 2008; received after revision 18 August 2008; accepted 22 August 2008     

Signs, toy models, and the a priori: from Helmholtz to Wittgenstein

The Marburg neo-Kantians argue that Hermann von Helmholtz’s empiricist account of the a priori does not account for certain knowledge, since it is based on a psychological phenomenon, trust in the regularities of nature. They argue that Helmholtz’s account raises the ‘problem of validity’ (Gültigkeitsproblem): how to establish a warranted claim that observed regularities are based on actual relations. I reconstruct Heinrich Hertz’s and Ludwig Wittgenstein’s Bild theoretic answer to the problem of validity: that scientists and philosophers can depict the necessary a priori constraints on states of affairs in a given system, and can establish whether these relations are actual relations in nature. The analysis of necessity within a system is a lasting contribution of the Bild theory. However, Hertz and Wittgenstein argue that the logical and mathematical sentences of a Bild are rules, tools for constructing relations, and the rules themselves are meaningless outside the theory. Carnap revises the argument for validity by attempting to give semantic rules for translation between frameworks. Russell and Quine object that pragmatics better accounts for the role of a priori reasoning in translating between frameworks. The conclusion of the tale, then, is a partial vindication of Helmholtz’s original account.     

Identification of 2-deoxyecdysone from the mite,Tyrophagus putrescentiae

2-Deoxyecdysone was purified from the miteTyrophagus putrescentiae and monitored by a radioimmunoassay. The compound was identified by GC-MS and LC-MS. This is the first report of an endogenous ecdysteroid from a mite.     

Influence of environmental conditions on the amount of N2O released from activated sludge in a domestic waste water treatment plant

Waste water purification is characterized by intensive mineralization and nitrification processes. Because of the high O₂ demand, temporarily anaerobic conditions may be produced, and denitrification by nitrifying organisms as well as heterotropic denitrification may contribute to N₂O release. In situ measurements (1993–1994) suggest that N₂O is released from activated sludge in a domestic waste water treatment plant at an average rate of 1040 g m^–2h^–1 with a range between zero and 6198 g m^–2h^–1. The production of N₂O seems to be related to the concentration of NO ₂ ^– and NO ₃ ^– as well as to the pH. In the waste water about 75–200 g N₂O l^–1 is dissolved. This N₂O is released after discharge into the receiving waters. The N₂O is produced essentially by nitrification rather than by heterotropic denitrification. On a long-term scale the increasing use of mechanical-biological waste water purification plants world-wide may add increasingly to the anthropogenic production of N₂O, although the present amount of N₂O produced is negligible compared to its global terrestrial production.     

Molecular ecology of a Neolithic meadow: The DNA of the grass remains from the archaeological site of the Tyrolean Iceman

The paper reports on the molecular analysis of samples of approximately 5,300-year-old grass found at the alpine archaeological site where the so-called Tyrolean Iceman was discovered. The grass comes from a cloak made of long grass blades and/or the stuffing of the snow footwear worn by the Iceman. The results show that while the largest fraction of the DNA extractable from the grass is of foreign origin, a much smaller part belongs to the original genetic material of the grass itself, and can be used as a valuable taxonomic clue to the plant species utilized by neolithic men to manufacture their equipment. On the other hand, the foreign DNA, or at least a portion of it, comes from microorganisms-mainly filamentous fungi and unicellular algae-which seem to have been associated with the grass since the time the grass was harvested.