双管基因枪介导的基因瞬时表达技术在拟南芥中的应用

基因瞬时表达是植物中研究目标基因功能的常用手段。在模式植物拟南芥(Arabidopsis thaliana)中, 相比原生质体和农杆菌介导的基因异源表达技术, 利用粒子轰击进行基因瞬时表达一直鲜有报道。其主要原因是拟南芥叶型相对较小、基因枪操作相对烦琐以及基因表达效率差异较大。该研究通过优化双管基因枪系统, 在营养生长旺盛的拟南芥莲座叶中实现GFP和GUS基因高效表达。同时, 通过GUS报告基因明确了坏死诱导因子BAX、Avh238和ATR13/Rpp13激发拟南芥细胞坏死的表型。但在本氏烟(Nicotiana benthamiana)中明显诱导细胞坏死的Avrblb1/RB基因对, 在拟南芥中却丧失了诱导细胞坏死的活性。由于双管基因枪系统每次轰击时设置平行对照, 可有效降低转化实验中的样本变异度, 为拟南芥及其突变体研究中准确评价基因功能和高通量筛选目标基因提供新的技术参考。     

Locomotor performance in an invasive species: cane toads from the invasion front have greater endurance, but not speed, compared to conspecifics from a long-colonised area

Cane toads (Bufo marinus) are now moving about 5 times faster through tropical Australia than they did a half-century ago, during the early phases of toad invasion. Radio-tracking has revealed higher daily rates of displacement by toads at the invasion front compared to those from long-colonised areas: toads from frontal populations follow straighter paths, move more often, and move further per displacement than do toads from older (long-established) populations. Are these higher movement rates of invasion-front toads associated with modified locomotor performance (e.g. speed, endurance)? In an outdoor raceway, toads collected from the invasion front had similar speeds, but threefold greater endurance, compared to conspecifics collected from a long-established population. Thus, increased daily displacement in invasion-front toads does not appear to be driven by changes in locomotor speed. Instead, increased dispersal is associated with higher endurance, suggesting that invasion-front toads tend to spend more time moving than do their less dispersive conspecifics. Whether this increased endurance is a cause or consequence of behavioural shifts associated with rapid dispersal is unclear. Nonetheless, shifts in endurance between frontal and core populations of this invasive species point to the complex panoply of traits affected by selection for increased dispersal ability on expanding population fronts.     

Novel ATP-dependent calcium transport component from rat liver plasma membranes. The transporter and the previously reported (Ca2+-Mg2+)-ATPase are different proteins

An ATP-dependent calcium transport component from rat liver plasma membranes was solubilized by cholate and reconstituted into egg lecithin vesicles by a cholate dialysis procedure. The uptake of Ca2+ into the reconstituted vesicles was ATP-dependent and the trapped Ca2+ could be released by A23187. Nucleotides, including ADP, UTP, GTP, CTP, GDP, AMP, and adenyl-5'-yl beta, gamma-imidophosphate, and p-nitrophenylphosphate did not substitute for ATP. The concentration of ATP required for half-maximal stimulation of Ca2+ uptake into the reconstituted vesicles was 6.2 microM. Magnesium was required for calcium uptake. Inhibitors of mitochondrial calcium-sequestering activities, i.e. oligomycin, sodium azide, ruthenium red, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and valinomycin did not affect the uptake of Ca2+ into the vesicles. In addition, strophanthidin and p-chloromercuribenzoate did not affect the transport. Calcium transport, however, was inhibited by vanadate in a concentration-dependent fashion with a K0.5 of 10 microM. A calcium-stimulated, vanadate-inhibitable phosphoprotein was demonstrated in the reconstituted vesicles with an apparent molecular weight of 118,000 +/- 1,300. These properties of Ca2+ transport by vesicles reconstituted from liver plasma membranes suggest that this ATP-dependent Ca2+ transport component is different from the high affinity (Ca2+-Mg2+)-ATPase found in the same membrane preparation (Lotersztajn, S., Hanoune, J. and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215; Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020). When the entire reconstituted vesicle population was treated with ATP and 45Ca in a buffer containing oxalate, the vesicles with Ca2+ transport activity could be separated from other vesicles by centrifugation in a density gradient and the ATP-dependent Ca2+ transport component was purified approximately 9-fold. This indicates that transport-specific fractionation may be used to isolate the ATP-dependent Ca2+ transport component from liver plasma membrane.     

Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng

Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The K_m and V_max values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC₅₀ values were 16.00 μM and 9.83 μM, and K_i values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.     

Design of high performance nanozymes: a single-atom strategy

<正>Nanozymes, nanomaterials with enzyme-like characteristics,are emerging as novel artificial enzymes (Gao et al., 2007;Manea et al., 2004; Yan, 2018). They are superior to natural enzymes in many ways, such as higher stability, lower cost in preparation, and better robustness toward harsh environments (Wei and Wang, 2013). Various nanomaterials (e.g.,     

Role of superoxide in radiation-killing of Escherichia coli and in thymine release from thymidine

The role of superoxide and hydroxyl radicals in gamma-radiation-killing of Escherichia coli K12 was studied in aerated suspensions supplemented with formate, phosphate, superoxide dismutase, catalase and saturated with nitrous oxide. Nitrous oxide, which converts e-aq to .OH, caused decreased radiosensitivity. On the other hand, formate, which results in conversion of .OH to .O2-, resulted in an increased radiosensitivity. The results implicated .O2- as a major cause of radiation-mediated cell-killing. The addition of the enzymes, superoxide dismutase or catalase to the E. coli suspensions prior to and during irradiation had no effect on cell survival, indicating that the biologically significant site of generation and action of .O2- is an intracellular one. Further studies were undertaken to examine the role of superoxide in DNA damage. The release of thymine from the DNA base, thymidine was studied as a result of gamma-irradiation and of chemically generated superoxide (using KO2 in dimethyl sulfoxide). Thymine was identified by HPLC and mass spectrometry. C-13 NMR analysis of the reaction mixture of thymidine with KO2 in dimethyl sulfoxide provided evidence for attack of .O2 at the ribosyl Cl' atom.     

Albino inflorescence proliferation of Dendrocalamus latiflorus

Summary Inflorescence proliferation is a plant tissue culture technique that, can be used to obtain in vitro inflorescences year-round without the intervening development of vegetative organs. In this study, we used albino mutant inflorescences of Dendrocalamus latiflorus as the original explant material to investigate, the effect of plant growth regulators on long-term inflorescence proliferation. The albino inflorescences proliferated on solidified Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ), and the optimal concentration for successful long-term inflorescence proliferation was 0.45 μM TDZ. A combination of α-naphthaleneacetic acid (NAA) with 0.45 μM TDZ inhibited the inflorescence proliferation. Inflorescences cultured on a TDZ-free medium supplemented with 26.82 μM NAA rooted in 21 d, vegetative shoots formed by 42 d and, in one case, flowering occurred after 63 d. The auxins 2,4-dichlorophenoxyacetic acid (2,4-D, 4.52 μM) and pieloram (4.14 μM) induced shoot formation. The protocol described can be used to produce large numbers of mutant inflorescences within a relatively short period of time.     

Evidence for the existence of three or more slow phases in the refolding of ribonuclease A and some characteristics of the phases

The slow refolding kinetics of RNase A have been analyzed, by using a nonlinear least-squares program for deconvoluting the kinetic phases and applying statistical tests for quality of fit. It is found that a minimum of three slow phases are required to fit the kinetic data properly, and this is true whether the method of detection is absorbance of fluorescence. Since the number of phases and the relaxation times for each phase are independent of the method of detection, it is concluded that the same three rate-limiting processes are seen by absorbance and fluorescence. These phases correspond to the XY, CT, and ct phases described in our earlier studies. The fact that fluorescence-detected kinetics are somewhat slower than absorbance-detected kinetics is a trivial effect due not to differences in relaxation times but to the fact that the amplitude of the CT phase is enhanced in fluorescence measurements, at the expense of the faster XY phase, because of intrinsic fluorescence changes associated with the isomerization of proline-93. By use of a new double-jump technique [Schmid, F.X., Grafl, R., Wrba, A., & Beintema, J.J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 872], it is shown that proline-93 isomerizes as the rate-limiting step in only one of the three phases, the CT phase, and that this phase involves only 25-30% of the RNase molecules. There is still no indication as to the molecular events that occur in the large, ammonium sulfate dependent XY phase, which is the pathway for formation of the nativelike intermediate.     

CircRNA has_circ_0006427 suppresses the progression of lung adenocarcinoma by regulating miR-6783–3p/DKK1 axis and inactivating Wnt/β-catenin signaling pathway

Recently, circular RNAs (circRNAs) are identified as a novel class of noncoding RNAs playing important roles in human malignant tumors. However, the regulatory function of circRNA in lung adenocarcinoma (LUAD) is still largely unknown. Present study aimed to explore the role of circ_0006427 in LUAD progression. Firstly, the downregulation of circ_0006427 in LUAD tissues and cell lines was revealed by microarray analysis and qRT-PCR analysis. And we also confirmed the circ_0006427 as a prognostic target in LUAD patients. Functionally, overexpression of circ_0006427 effectively suppressed cell proliferation, migration and invasion. Mechanistically, circ_0006427 was found to be predominantly located in the cytoplasm of LUCA cell, and was further revealed to positively regulate DKK1 in LUAD by sponging miR-6783–3p. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and western blot analysis revealed that circ_0006427 inactivated Wnt/β-catenin signaling pathway by upregulating DKK1. At last, rescue assays proved the function of circ_0006427/miR-6783–3p/DKK1 axis in LUAD progression. In conclusion, our study revealed that circ_0006427 suppressed lung adenocarcinoma progression through regulating miR-6783–3p/DKK1 axis.     

Hydrolysis of dansyl-peptide substrates by leucine aminopeptidase: origin of dansyl fluorescence changes during hydrolysis

The origin of the fluorescence changes observed in stopped-flow experiments of the hydrolysis of three 5-(dimethylamino)naphthalene-1-sulfonyl-(dansyl) peptide substrates by porcine kidney cytosol leucine aminopeptidase has been investigated. The substrates used all have the potential to accept energy from aromatic residues of the enzyme via resonance energy transfer when they are bound as enzyme-substrate complexes, indicating that fluorescence changes due to the buildup and decay of such intermediates are possible. However, the fluorescence of these substrates differs from that of the products, and direct excitation of their dansyl groups during hydrolysis can also be responsible for the observed fluorescence changes due to changes in the concentrations of free substrate and product. The dansyl fluorescence changes observed with excitation wavelengths near 280 nm are not accompanied by quenching of the enzyme fluorescence, as would be expected if there were enzyme-to-substrate energy transfer. The magnitude of the maximal fluorescence change at a fixed concentration of substrate is also independent of the enzyme concentration. Furthermore, the excitation profile for the fluorescence changes shows that they arise from direct excitation of the dansyl group. Thus, there is no energy transfer in these reactions, and the fluorescence changes observed arise from direct excitation of the dansyl group and reflect the instantaneous concentration of substrate. This behavior contrasts sharply with that for the reaction of carboxypeptidase A with dansyl-Gly-Tyr, which has been studied as a positive control for an energy-transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)