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991.
Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. PTP inhibitors provide potential treatment of human diseases/conditions such as diabetes and obesity as well as useful tools for studying the function of PTPs in signaling pathways. In this work, we have shown that certain aryl-substituted aldehydes act as reversible, slow-binding inhibitors of modest potency against PTP1B, SHP-1, and a dual-specificity phosphatase, VHR. Attachment of the tripeptide Gly-Glu-Glu to the para position of cinnamaldehyde resulted in an inhibitor (Cinn-GEE) of substantially increased potency against all three enzymes (e.g., K(I) = 5.4 microM against PTP1B). The mechanism of inhibition was investigated using Cinn-GEE specifically labeled with (13)C at the aldehyde carbon and (1)H-(13)C heteronuclear single-quantum coherence spectroscopy. While Cinn-GEE alone showed a single cross-peak at delta 9.64 ((1)H) and delta 201 ((13)C), the PTP1B/Cinn-GEE complex showed three distinct cross-peaks at delta 7.6-7.8 ((1)H) and 130-137 ((13)C). Mutation of the catalytic cysteine (Cys-215 in PTP1B) into alanine had no effect on the cross-peaks, whereas mutation of a conserved active-site arginine (Arg-221 in PTP1B) to alanine abolished all three cross-peaks. Similar experiments with Cinn-GEE that had been labeled with (13)C at the benzylic position revealed a change in the hybridization state (from sp(2) to sp(3)) for the benzylic carbon as a result of binding to PTP1B. These results rule out the possibility of a free aldehyde, aldehyde hydrate, or hemithioacetal as the enzyme-bound inhibitor form. Instead, the data are consistent with the formation of an enamine between the aldehyde group of the inhibitor and the guanidine group of Arg-221 in the PTP1B active site. These aldehydes may provide a general core structure that can be further developed into highly potent and specific PTP inhibitors. 相似文献
992.
t-PA突变体工程细胞株FSGGI48形态与其亲代细胞CHO-dhfr相似呈多角形,类似上皮细胞。在MTX加压至5×10~(-6)mol/L时,少数细胞形态略变瘦长,但仍呈多角形,因此该工程细胞株形态正常。抗体中和抑制试验及纤维蛋白板80℃加热试验显示,FSGGI48细胞表达产物与st-PA的活性均能被抗t-PA抗体所抑制,而胰酶的活性则不被抑制,且二者在80℃加热的纤维蛋白板上均不产生溶解圈,胰酶则产生溶解圈,由此可知,该细胞株表达产物为特异的rt-PA产品。细胞无血清培养上清经FA-PA检测,亚克隆株表达水平为4000IU/10~6细胞/24h。分别测定冻存3个月后复苏和体外传代3个月以上细胞的表达水平,结果显示部分亚克隆细胞株表达水平下降,多数仍为3000-4000IU/10~6细胞/24h,说明细胞株稳定性好。裸鼠试验表明,该工程株活细胞、细胞DNA、纯化的细胞表达产物均无致瘤性。支原体检查结果为阴性。染色体分析显示,FSGGI48细胞与其亲代细胞(CHO-dhfr细胞)染色体数目相同均为20条,但均有不同程度不同类型的畸变。CHO-dhfr细胞畸变率为6%。该工程细胞的畸变率为15%-18%,在允许范围内。结果证明,t-PA突变体细胞株FSGGI48为性能优良的工程细胞株。 相似文献
993.
Effects of phosphite (Phi) on phosphate (Pi) starvation responses were determined in Ulva lactuca L. by incubation in Pi‐limited (1 μM NaH2PO4) or Pi‐sufficient (100 μM NaH2PO4) seawater containing 0–3 mM Phi. Exposure to 1 μM NaH2PO4 decreased the growth rate and the content of free Pi and esterified‐P but increased the activities of extracellular alkaline phosphatase (EC 3.1.2.1) and intracellular acid phosphatase (ACP; EC 3.1.2.2); two ACP isozymes observed by activity staining on isoelectric focussing (IEF) gel were induced. The Km value of Pi uptake rate was decreased by incubation with 1 μM NaH2PO4 and the decrease in Km value was inhibited by 2 mM Phi, reflecting the operation of a high‐affinity Pi uptake system at low Pi concentrations. In the presence of Phi, the growth rate of Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. As Phi concentrations were increased from 0 to 2 mM, the free Pi contents in both Pi‐sufficient and Pi‐starved thalli decreased, but the esterified‐P contents in Pi‐starved thalli increased, whereas those in Pi‐sufficient thalli increased at 1 mM Phi and decreased at 2 mM Phi. Cell wall localized AP activity in both Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. Intracellular ACP activity in Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM but was not affected in Pi‐sufficient thalli. The induction of ACP isozyme activity and high‐affinity Pi uptake system in Pi‐starved thalli was inhibited by Phi. The present investigation shows that Phi interrupts the sensing mechanisms of U. lactuca to Pi‐limiting conditions. 相似文献
994.
The Kucong, a branch of Lahu and an indigenous ethnic group living in Jinping County, Yunnan Province, China, have a very short history of settled village life (about 40 years). According to the data of ethnobotanical investigation, 118 species of medicinal plants belonging to 57 families and 98 genera used by Lahu healers have been inventoried. The characteristics of medicinal plants coming from a great number of families and genera relate to the traditional life-style of the Lahu. The more common the diseases are, the more abundant the related medicinal knowledge is. The most frequently used parts of medicinal plants are the leaf (35.22%) and root (32.08%). The disappearance of environments related to the traditional life-style of the Lahu threatens the existence of their traditional medicinal knowledge, similar to threats facing many other traditional medicines. Collection and inventory should be essential research fields in studies of traditional medicine. 相似文献
995.
人胎盘c-fms胞外功能区基因片段的扩增 总被引:1,自引:0,他引:1
人胎盘c┐fms胞外功能区基因片段的扩增*王昕蔡辉国陈佩珍吴克复郑德先(中国医学科学院,中国协和医科大学血液学研究所,天津300020)Amplificationofc┐fmsExtracelularDomainfromHumanPlacentaWa... 相似文献
996.
重组抗体—尿激酶导向溶栓剂的基因构建及表达 总被引:5,自引:0,他引:5
为了获得高效、高特异性溶栓药物,应用基因工程技术,成功的表达了由人源化抗人活化血小板单抗和单链尿酶组成的抗体导向溶栓剂(SZ51Hu-scuPA)。通过基因重组PCR方法将scuPA全长cDNA的N末端连接在SZ51重链恒区CH3末端,构建了含有目的蛋白融合基因的真核表达载体αlys30-SZ51VH/Hu-scuPA。采用脂转染法将表达载体导入分泌SZ51VK/Hu轻链的小鼠骨髓瘤细胞中,筛选出 相似文献
997.
Gary N. Parkinson Youling Wu Pei Fan Joachim Kohn Jean Baum Helen M. Berman 《Biopolymers》1994,34(3):403-414
Urethane bonds, derived from the hydroxyl group of the tyrosine side chain, have been investigated as a new type of amide bond mimetic in the design of pseudopeptides. The structure of a representative cyclic pseudotetrapeptide that consists of an — Ala — Tyr(urethane) Ala — Tyr (urethane) sequence fused into a rigid ring has been studied in the solid state by x-ray crystallography and in solution by two-dimensional nmr techniques. The cyclic pseudotetrapeptide has an oblong shape. The backbone urethane bonds assume a trans–trans conformation. The carbonyl groups in the ring have an alternating pattern of down, up, down, up with respect to the average ring plane. Solution nmr studies give observed nuclear Overhauser effects and coupling constants largely in agreement with the crystal structure. However, in solution the observed structure is likely to be conformationally averaged, and in the averaged structure, the urethane bond is perpendicular to the plane of the aromatic ring of the tyrosine, while in the crystal it is close to this plane. These differences may be explained by intermolecular hydrogen-bonding interactions. Four aspects of the conformation of the cyclic pseudotetrapeptide were investigated in detail: the tyrosine residue with the attached side-chain urethane bond (the tyrosine-urethane unit), the conformation of the two urethane backbone linkages, the conformation of the two conventional peptide bonds within this unusual ring structure, and the tight turns within the cyclic pseudotetrapeptide. The conformation of the tight turns present in the cyclic pseudotetrapeptide is very similar to that of a β-bend of type II. Intermolecular hydrogen bonding, joining adjacent layers of the cyclic pseudotetrapeptide in the solid state, resemble a parallel β-pleated sheet. The presence of these structural motifs in the cyclic pseudotetrapeptide indicates that the tyrosine urethane unit may find applications in peptide and protein engineering. © 1994 John Wiley & Sons, Inc. 相似文献
998.
999.
本文以早熟品种冀麦31号和晚熟品系88—4284萌动种子为材料,报道了等离子束处理对小麦种子萌发的影响和细胞学效应。观察表明,等离子束长时间处理抑制种子发芽。在根尖细胞有丝分裂中期观察到染色体断裂和断片,冀麦31号染色体断裂的频率为0.49—1.34%,88—4284为0.21—2.14%。随着处理时间延长,染色体断裂频率逐渐提高。有丝分裂后期和末期出现大量的落后染色单体和染色体桥。在花粉母细胞减数分裂中期Ⅰ,经过等离子束处理的材料出现环状单价体。后期Ⅰ观察到染色体倒位造成的染色体桥和断片。在四分体期还有微核出现。 相似文献
1000.
Soil microbial respiration is expected to show adaptations to changing temperatures, greatly weakening the magnitude of feedback over time, as shown in labile carbon substrates. However, whether such thermal adaptation persists during long-term soil carbon decomposition as carbon substrates decrease in decomposability remains unknown. Here, we conducted a 6-year incubation experiment in natural and arable soils with distinct properties under three temperatures (10, 20 and 30°C). Mass-specific microbial respiration was consistently lower under higher long-term incubation temperatures, suggesting the occurrence and persistence of microbial thermal adaptation in long-term soil carbon decomposition. Furthermore, changes in microbial community composition and function largely explained the persistence of microbial respiratory thermal adaptation. If such thermal adaptation generally occurs in large low-decomposability carbon pools, warming-induced soil carbon losses may be lower than previously predicted and thus may not contribute as much as expected to greenhouse warming. 相似文献