ocs/mas|一个受损伤和植物激素诱导的嵌合启动子的构建与功能分析

选择适宜的转录调控序列以提高启动子的转录效率,增强外源基因在转基因植株中的表达,对改良作物的抗病虫性具有重要意义。将甘露碱合成酶基因(mas)启动子和章鱼碱合成酶基因(ocs)增强子杂合而成的嵌合启动子ocs/mas与GUS报告基因连接,构建了植物表达载体pOMS-GUS。对照载体pMAS-GUS仅携带mas启动子驱动的GUS基因。利用根癌农杆菌介导法,将以上植物表达载体分别转化烟草。应用半定量RT-PCR和GUS荧光定量分析法分别检测不同胁迫条件下启动子驱动的GUS基因表达量的变化。结果显示,未诱导处理的转基因植株GUS基因仅有微弱表达。伤害处理1h后,mas启动子驱动的GUS活性是未诱导处理的1.8倍,而嵌合启动子ocs/mas的诱导表达活性是未处理的5.7倍。植物激素水杨酸(SA)和茉莉酸甲酯(MJ)处理也诱导了较高水平的ocs/mas嵌合启动子活性;而且SA和MJ联合作用时呈现叠加效应,转基因烟草的GUS活性明显高于伤害处理后的GUS表达水平。以上结果表明,ocs/mas嵌合启动子是一种强诱导型启动子,可以接受多种刺激因子的诱导,从而为更有效地改良作物抗病虫的能力提供新的候选高效启动子元件。     

Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase

Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex.     

FXYD proteins stabilize Na,K-ATPase: amplification of specific phosphatidylserine-protein interactions

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1β1) to form α1β1FXYD complexes. Compared with the control (α1β1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C₁₂E₈), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E₁ ↔ E₂ conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1β1 or α1β1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.     

Defining a process operating window for the synthesis of 5-methyluridine by transglycosylation of guanosine and thymine

This paper describes a high yielding coupled enzymatic reaction using Bacillus halodurans purine nucleoside phosphorylase (PNP) and E. coli uridine phosphorylase (UP) for synthesis of 5-methyluridine (5-MU) by transglycosylation. Key parameters such as reaction temperature, pH, reactant loading, reactor configuration and enzyme loading were investigated. A guanosine conversion of 95% and a 5-MU yield of 85% were achieved at 1 l scale, with a productivity of 10 g l⁻¹ h⁻¹.     

Ruminal transport and metabolism of short-chain fatty acids (SCFA) in vitro: effect of SCFA chain length and pH

The unidirectional transport and metabolism of 14C-labeled acetate, propionate and butyrate across the isolated bovine rumen epithelium was measured in vitro by the Ussing chamber technique. There was a significant, but relatively small, net secretion of acetate and propionate, and a large and significant net absorption of butyrate. The results demonstrate that the mucosal-serosal (MS) pathway for short-chain fatty acids (SCFA) is different from the serosal-mucosal (SM) pathway, and that butyrate is treated differently from acetate and propionate by the epithelium. The results support that the main route for epithelial SCFA transport is transcellular. The correlation between SCFA lipophility and the flux rate was positive but weak at both pH 7.3 and 6.0. Decreasing pH increased all SCFA fluxes significantly, but not proportionally to the increase of protonized SCFA in the bathing solution. There was a significant and apparently non-competitive interaction between the transport of acetate, propionate and butyrate. It seems that mediated transport mechanisms must be involved in epithelial SCFA transport in the bovine rumen, but the data do not exclude that passive diffusion could account for a significant part of the flux. The metabolism of SCFA in the Ussing chamber system was considerable, and there was a clear preference for excretion of CO2 from this metabolism to the mucosal side, while side preference for non-CO2 metabolite excretion was not studied. Of the propionate and butyrate transported in the MS direction, 78 and 95% was metabolised, while only 37 and 38% was metabolised in the SM direction (acetate metabolism could not be measured). There was, however, no simple relation between the degree of metabolism and the transport rate or the transport asymmetry of the SCFA.     

Contribution of serum albumin to the transport of orally administered L-tryptophan into liver of rats with L-tryptophan depletion

Summary The role of serum albumin in the transport of orally administered L-tryptophan (Trp) into rat tissues was examined using analbuminemic and Sprague-Dawley (SD) rats with and without a-methyl-DL-tryptophan (AMT)-induced Trp depletion. Trp was orally administered to rats 16h after AMT or 0.85% NaCl administration, when liver tryptophan 2,3-dioxygenase and protein synthetic activities in AMT-treated rats were similar to those of 0.85% NaCl-treated rats. After oral Trp administration, regardless of the presence or absence of Trp depletion, free serum Trp concentrations were similar in the analbuminemic and SD rats, while total serum Trp concentrations were lower in analbuminemic rats than in SD rats. Although liver, brain, and muscle Trp concentrations after oral Trp administration under Trp depletion were lower in analbuminemic rats than in SD rats, the ratio of the liver Trp concentration in analbuminemic rats to that in SD rats was smaller than that of the brain or muscle Trp concentration. These results suggest that variations in serum albumin levels could affect the transport of orally administered Trp into the liver of rats with Trp depletion.     

Effects of Methylphenidate Analogues on Phenethylamine Substrates for the Striatal Dopamine Transporter

Methylphenidate (MPD) was found to inhibit competitively the striatal dopamine transporter (DAT) and bind at sites on the DAT in common with both cocaine (a non-substrate site ligand) and amphetamine (a substrate site ligand). Some methylphenidate analogues modified on the aromatic ring and/or at the nitrogen were tested to determine whether the profile of inhibition could be altered. None was found to stimulate the release of dopamine in the time frame (< or = 60 s) of the experiments conducted, and each of the analogues tested was found to noncompetitively inhibit the transport of dopamine. It was found that halogenating the aromatic ring with chlorine (threo-3,4-dichloromethylphenidate hydrochloride; compound 1) increased the affinity of MPD to inhibit the transport of dopamine. A derivative of MPD with simultaneous, single methyl group substitutions on the phenyl ring and at the nitrogen (threo-N-methyl-4-methylphenidate hydrochloride; compound 2) bound at a site in common with MPD. A benzyl group positioned at the nitrogen (threo-N-benzylmethylphenidate hydrochloride; compound 3) imparted properties to the inhibitor in which binding at substrate and non-substrate sites could be distinguished. This analogue bound at a mutually interacting site with that of methylphenidate and had a K(int) value of 4.29 microM. Furthermore, the N-substituted analogues (compounds 2 and 3), although clearly inhibitors of dopamine transport, were found to attenuate dramatically the inhibition of dopamine transport by amphetamine, suggesting that the development of an antagonist for substrate analogue drugs of abuse may be possible.     

Expression, Isolation, and Crystallization of the Catalytic Domain of CopB, a Putative Copper Transporting ATPase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

The P-type CPX-ATPases are responsible for the transport of heavy metal ions in archaea, bacteria, and eukaryotes. We have chosen one of the two CPX-ATPases of the thermophile Sulfolobus solfataricus, CopB (= SSO2896) for the investigation of the molecular mechanism of this integral membrane protein. We recombinately expressed three different soluble domains of this protein (named CopB-A, CopB-B, and CopB-C) in Escherichia coli and purified them to homogeneity. 3D crystals of CopB-B, the 29 kDa catalytic ATP binding/phosphorylation domain were produced, which diffracted to a resolution of 2.2 A. CopB-B has heavy metal stimulated phosphatase activity, which was half maximal in the presence of 80 microM Cu2+. The protein forms a phosphorylated intermediate with the substrate gamma-(32P)-ATP. No specific activation of the polypeptide was observed, when CopB-B phosphatase activity was tested in the presence of the purified CopB-C and CopB-A proteins, which provide the cation binding and the phosphatase domains. We conclude that CopB is a putatively copper translocating ATPase, in which structural elements integrally located in the membrane are required for full, coordinated activation of the catalytic ATP binding domain.     

A transporter of<Emphasis Type="Italic"> Escherichia coli</Emphasis> specific for<Emphasis Type="SmallCaps"> l</Emphasis>- and<Emphasis Type="SmallCaps"> d</Emphasis>-methionine is the prototype for a new family within the ABC superfamily

An ABC-type transporter in Escherichia coli that transports both l- and d-methionine, but not other natural amino acids, was identified. This system is the first functionally characterized member of a novel family of bacterial permeases within the ABC superfamily. This family was designated the methionine uptake transporter (MUT) family (TC #3.A.1.23). The proteins that comprise the transporters of this family were analyzed phylogenetically, revealing the probable existence of several sequence-divergent primordial paralogues, no more than two of which have been transmitted to any currently sequenced organism. In addition, MetJ, the pleiotropic methionine repressor protein, was shown to negatively control expression of the operon encoding the ABC-type methionine uptake system. The identification of MetJ binding sites (in gram-negative bacteria) or S-boxes (in gram-positive bacteria) in the promoter regions of several MUT transporter-encoding operons suggests that many MUT family members transport organic sulfur compounds. Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.