A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Stimulation of fission yeast and mouse Hop2-Mnd1 of the Dmc1 and Rad51 recombinases

Genetic analysis of fission yeast suggests a role for the spHop2–Mnd1 proteins in the Rad51 and Dmc1-dependent meiotic recombination pathways. In order to gain biochemical insights into this process, we purified Schizosaccharomyces pombe Hop2-Mnd1 to homogeneity. spHop2 and spMnd1 interact by co-immunoprecipitation and two-hybrid analysis. Electron microscopy reveals that S. pombe Hop2–Mnd1 binds single-strand DNA ends of 3′-tailed DNA. Interestingly, spHop2-Mnd1 promotes the renaturation of complementary single-strand DNA and catalyses strand exchange reactions with short oligonucleotides. Importantly, we show that spHop2-Mnd1 stimulates spDmc1-dependent strand exchange and strand invasion. Ca²⁺ alleviate the requirement for the order of addition of the proteins on DNA. We also demonstrate that while spHop2-Mnd1 affects spDmc1 specifically, mHop2 or mHop2-Mnd1 stimulates both the hRad51 and hDmc1 recombinases in strand exchange assays. Thus, our results suggest a crucial role for S. pombe and mouse Hop2-Mnd1 in homologous pairing and strand exchange and reveal evolutionary divergence in their specificity for the Dmc1 and Rad51 recombinases.     

每搏量变异度在围手术期容量治疗监测中的应用

每搏量变异度是动态的容量监测指标.机械通气患者心肺的相互作用是每搏量变异度的产生基础,通过动脉压力波形分析技术可以进行连续监测.每搏量变异度能够准确预测容量治疗反应,与静态的血流动力学参数相比,对于优化心输出量和组织氧供更有优势,但也存在一定的局限性.每搏量变异度受多种因素影响且不能用于自主呼吸和心律失常的患者.临床应用时应该综合考虑其影响因素,结合其他的指标和方法指导容量治疗.     

Generation of supercoils in nicked and gapped DNA drives DNA unknotting and postreplicative decatenation

Due to the helical structure of DNA the process of DNA replication is topologically complex. Freshly replicated DNA molecules are catenated with each other and are frequently knotted. For proper functioning of DNA it is necessary to remove all of these entanglements. This is done by DNA topoisomerases that pass DNA segments through each other. However, it has been a riddle how DNA topoisomerases select the sites of their action. In highly crowded DNA in living cells random passages between contacting segments would only increase the extent of entanglement. Using molecular dynamics simulations we observed that in actively supercoiled DNA molecules the entanglements resulting from DNA knotting or catenation spontaneously approach sites of nicks and gaps in the DNA. Type I topoisomerases, that preferentially act at sites of nick and gaps, are thus naturally provided with DNA–DNA juxtapositions where a passage results in an error-free DNA unknotting or DNA decatenation.     

Plasticization of poly(L-lactide) with poly(propylene glycol)

A new plasticizer for poly(L-lactide) (PLA)-poly(propylene glycol) (PPG) is proposed. The advantage of using PPG is that it does not crystallize, has low glass transition temperature, and is miscible with PLA. PLA was plasticized with PPGs with nominal Mw of 425 and 1000 g/mol. Poly(ethylene glycol) (PEG), long known as a plasticizer for PLA, with nominal Mw of 600 g/mol, was also used to plasticize PLA for comparison. The thermal and tensile properties of PLA and PLA with 5-12.5 wt % of the plasticizers were studied. In blends of PLA with PPGs the glass transition temperature was lower than that of neat PLA. Both PPGs enhanced the crystallizability of PLA albeit less than PEG. All of the plasticizers increased also the ability of PLA to plastic deformation which was reflected in a decrease of yield stress and in an increase of elongation at break. The effect was enhanced by the higher PPG content and also by lower molecular weight of PPG. A phase separation occurred only in the blend containing 12.5 wt % of PPG with higher molecular weight. The evidences of crazing were found in deformed samples of PLA with low plasticizer content, whereas the samples with higher content of plasticizers crystallized due to deformation.     

Cyclic Expression of A Nuclear Protein In A Dinoflagellate

Nuclei of the dinoflagellate Crypthecodinium cohnii strain Whd were isolated and nuclear proteins were extracted in three fractions, corresponding to the increasing affinity of these proteins to genomic DNA. One fraction contained two major bands (48- and 46-kDa) and antibodies specific to this fraction revealed two major bands by Western blot on nuclear extracts, corresponding to the 46- and 48-kDa bands. The 48-kDa protein was detected in G1 phase but not in M phase cells. An expression cDNA library of C. cohnii was screened with these antibodies, and two different open reading frames were isolated. Dinoflagellate nuclear associated protein (Dinap1), one of these coding sequences, was produced in E. coli and appeared to correspond to the 48-kDa nuclear protein. No homologue of this sequence was found in the data bases, but two regions were identified, one including two putative zinc finger repeats, and one coding for two potential W/W domains. The second coding sequence showed a low similarity to non-specific sterol carrier proteins. Immunocytolocalization with specific polyclonal antibodies to recombinant Dinap1 showed that the nucleus was immunoreactive only during the G1 phase: the nucleoplasm was immunostained, while chromosome cores and nuclear envelopes were negative.