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1.
We have analyzed the cell wall of the radio-resistant bacterium Deinococcus radiodurans. Unexpectedly, the bacterial envelope appears to be organized in different complexes of high molecular weight. Each complex is composed of several proteins, most of which are coded by genes of unknown function and the majority are constituents of the inner/outer membrane system. One of the most abundant complexes is constituted by the gene DR_0774. This protein is a type of secretin which is a known subunit of the homo-oligomeric channel that represents the main bulk of the type IV piliation family. Finally, a minor component of the pink envelope consists of several inner-membrane proteins. The implications of these findings are discussed.  相似文献   
2.
The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin–angiotensin system. (P)RR is known to form a homodimer, but the region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD participates in dimerization.  相似文献   
3.
The human Z-type α1-antitrypsin variant has a strong tendency to accumulate folding intermediates due to extremely slow protein folding within the endoplasmic reticulum (ER) of hepatocytes. Human α1-antitrypsin has 17 peptidyl-prolyl bonds per molecule; thus, the effect of peptidyl-prolyl isomerases on Z-type α1-antitrypsin protein folding was analyzed in this study. The protein level of Cpr2p, a yeast ER peptidyl-prolyl isomerase, increased more than two-fold in Z-type α1-antitrypsin-expressing yeast cells compared to that in wild-type α1-antitrypsin-expressing cells. When CPR2 was deleted from the yeast genome, the cytotoxicity of Z-type α1-antitrypsin increased significantly. The interaction between Z-type α1-antitrypsin and Cpr2p was confirmed by co-immunoprecipitation. In vitro folding assays showed that Cpr2p facilitated Z-type α1-antitrypsin folding into the native state. Furthermore, Cpr2p overexpression significantly increased the extracellular secretion of Z-type α1-antitrypsin. Our results indicate that ER peptidyl-prolyl isomerases may rescue Z-type α1-antitrypsin molecules from retarded folding and eventually relieve clinical symptoms caused by this pathological α1-antitrypsin.  相似文献   
4.
The effect of trifluoperazine (TFP) on the ATPase activity of soluble and paniculate F1ATPase and on ATP synthesis driven by succinate oxidation in submitochondrial particles from bovine heart was studied at pH 7.4 and 8.8. At the two pH. TFP inhibited ATP hydrolysis. Inorganic phosphate protected against the inhibiting action of TFP. The results on the effect of various concentrations of phosphate in the reversal of the action of TFP on hydrolysis at pH 7.4 and 8.8 showed that H2PO 4 is the species that competes with TFP. The effect of TFP on oxidative phosphorylation was studied at concentrations that do not produce uncoupling or affect the aerobic oxidation of succinate (<15M). TFP inhibited oxidative phosphorylation to a higher extent at pH 8.8 than at pH 7.4; this was through a diminution in theV max, and an increase in theK m for phosphate. Data on phosphate uptake during oxidative phosphorylation at several pH showed that H2PO 4 is the true substrate for oxidative phosphorylation. Thus, in both synthesis and hydrolysis of ATP, TFP and H2PO 4 interact with a common site. However, there is a difference in the sensitivity to TFP of ATP synthesis and hydrolysis; this is more noticeable at pH 8.8, i.e. ATPase activity of soluble F1 remains at about 40% of the activity of the control in a concentration range of TFP of 40–100M, whereas in oxidative phosphorylation 14M TFP produces a 60% inhibition of phosphate uptake.  相似文献   
5.
腺苷酸激酶基因在大肠杆菌中的可溶性高表达   总被引:6,自引:0,他引:6  
报道了鸡肌腺苷酸激酶基因的克隆和在温控启动子PRPL调控下在大肠杆菌中的可溶性高效表达.SDS-PAGE分析表明,鸡肌腺苷酸激酶的含量可占大肠杆菌细胞总蛋白含量的38%.利用Johnson等的干冰/乙醇-冰水浴反复冻融法,可将此重组蛋白进行富集,纯度可达85%以上.鸡肌腺苷酸激酶可与抗兔肌腺苷酸激酶单克隆抗体产生强的交叉反应.  相似文献   
6.

Background

AHSP is an erythroid molecular chaperone of the α-hemoglobin chains (α-Hb). Upon AHSP binding, native ferric α-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6th coordination bond with His(E7).

Methods

Recombinant AHSP, WT α-Hb:AHSP and α-HbHE7Q:AHSP complexes were expressed in Escherichia coli. Thermal denaturation curves were measured by circular dichroism for the isolated α-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of α-Hb bound to AHSP as well as α-Hb release from the α-Hb:AHSP complex were measured by time-resolved absorption spectroscopy.

Results

AHSP binding to α-Hb is kinetically controlled to prevail over direct binding with β-chains and is also thermodynamically controlled by the α-Hb redox state and not the liganded state of the ferrous α-Hb. The dramatic instability of isolated ferric α-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in α-HbH58(E7)Q:AHSP complex reduces the stabilizing effect of AHSP binding. Once the ferric α-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to α-Hb within the Hb-tetramer.

Conclusion

α-Hb reduction could trigger its release from AHSP toward its final Hb β-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis.

General significance

The cellular redox balance in Hb-synthesis should be considered as important as the relative proportional synthesis of both Hb-subunits and their heme cofactor. The in vivo role of AHSP is discussed in the context of the molecular disorders observed in thalassemia.  相似文献   
7.
Beta-N-oxalylamino-l-alanine (BOAA), a non-protein amino acid present in the seeds of Lathyrus Sativus (LS), is one of several neuroactive glutamate analogs reported to stimulate excitatory receptors and, in high concentrations, cause neuronal degeneration. In the present study, the in vivo acute effects of synthetic BOAA and LS seed extract were investigated on rat cerebellar cyclic GMP following intraperitoneal (10–100 mg/kg) or oral (100 mg/kg) administration of subconvulsive doses of toxin. Furthermore, the BOAA content in LS seeds and in the cerebellum of injected rats was determined by high performance liquid chromatograph analysis. A dose- and time-dependent increase of cerebellar cyclic guanosine monophosphate (cGMP) level was observed after intraperitoneal administration of synthetic BOAA or LS extract. The neurotoxin evoked a maximum stimulation 90 min after injection within the dose range of 50–75 mg/kg, elevating cGMP from basal levels of 5.3±0.5 pmol/mg protein to 15±1.3 pmol/mg protein. Similarly, the oral intake of LS-extracted neurotoxin resulted in the elevation of cGMP content. Kynurenic acid (300 mg/kg i.p.), a non specific excitatory amino acid antagonist, was effective in blocking LS BOAA-elicited cGMP enhancement. The data suggest that in the cerebellum acute administration of low concentrations of BOAA exert in vivo activation of glutamate receptors involved in the regulation of cGMP level.  相似文献   
8.
Seaweed extract, prepared by alkaline extraction of Ascophyllum nodosum (L.) Le Jol., applied either to the soil or to the foliage of tomato plants, produced leaves with higher chlorophyll levels than those of control plants. The effects on leaf chlorophyll content were investigated using a cucumber bioassay procedure devised for cytokinins. The seaweed extract was shown to increase the chlorophyll levels of the cucumber cotyledons, but ‘peaks’ of activity were obtained when widely different concentrations were used. The possibility that these effects were the result of betaines present in the extract was considered. Glycinebetaine, γ-aminobutyric acid betaine and δ-aminovaleric acid betaine all produced significantly enhanced chlorophyll concentrations in the cotyledons. ‘Peaks’ of activity were observed for each betaine: for glycinebetaine at 10−6 and between 10−4 and 101 mg 1−1, for γ-aminobutyric acid betaine at 10−6, between 10−4 and 10−1, and 101 mg 1−1, and for δ-aminovaleric acid betaine between 10−5 and 101 mg 1−1. It was concluded that the effects of enhancing chlorophyll levels produced by the seaweed extract were due, at least in part, to betaines.  相似文献   
9.
三裂叶野葛毛状根的诱导及其固体培养和液体培养   总被引:6,自引:1,他引:5  
发根农杆菌(Agrobacterium rhizogenes)ATCC15834感染三裂叶野葛(Pueraria phaseoloides)叶片外植体20 d后产生毛状根,毛状根可直接从叶片外植体叶脉处或从叶脉处产生的愈伤组织上产生。感染35d后,约85%的叶片外植体产生毛状根。毛状根能在无外源生长调节剂的 MS固体和液体培养基上自主生长。PCR扩增结果表明,发根农杆菌Ri质粒的rolBrolC基因已在三裂叶野葛毛状根基因组中整合并得到表达。与固体培养的毛状根相比,在液体培养基中培养的毛状根不仅生长迅速,也不会形成愈伤组织。在无外源生长调节剂的液体MS培养基中培养15d的三裂叶野葛毛状根的鲜重、干重、可溶性总糖含量及细胞内活性氧(ROS)含量分别为固体培养毛状根的1.59倍、1.18倍、5.25倍和1.16倍。  相似文献   
10.
用正交实验结果作方差分析表明:溶剂用量对试验结果有较显著的影响(a=0.10),提取温度对试验结果也有较大的影响,而提取时间对试验结果的影响不显著。喜树叶中水溶性糖的提取最优方案是A3C2B3,即溶剂用量为60ml,提取温度80℃,提取时间60min;喜树叶水溶性糖的提取最优方案是A2C2B1,即溶剂用量为40ml,提取温度80℃,提取时间20min。通过对喜树叶与枝的成对比较分析,叶与枝的水溶性糖含量无显著差异。  相似文献   
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