Solution structure of betacellulin,a new member of EGF-family ligands

The solution structure of the EGF-like domain of betacellulin (BTCe), a newly discovered member of the epidermal growth factor (EGF) family, has been determined using two-dimensional nuclear magnetic resonance spectroscopy. This is the first report to identify the solution structure of the EGF-family ligand monomers that interact with both ErbB-1 and ErbB-4. The solution structure of BTCe was calculated using 538 NMR-derived restraints. The overall structure of BTCe was stabilized by three disulfide bonds, a hydrophobic core, and 23 hydrogen bonds. It appears that BTCe is comprised of five beta-strands and one short 3(10) helical turn. The secondary structural elements of BTCe are basically similar to those of the other EGF-family proteins, except that several significant variations of the structural properties were found. It is suggested that the structural variations between BTCe and the other EGF-family ligands may affect the specific receptor-recognition properties of EGF-family ligands.     

Structure-function studies of mEGF: Probing the type Iβ-turn between residues 25 and 26

The interaction between epidermal growth factor (EGF) and its receptor molecule is not completely understood and has received much attention recently. Studies combining site-directed mutagenesis and NMR spectroscopy have identified a number of EGF residues that are required for activity and are believed to interact directly with the receptor. Instead of focusing on these residues, this study combines site-directed mutagenesis and NMR spectroscopy to probe the role of the type I-bend located between residues 25 and 26 of the N-terminal subdomain of the protein. Ser25 of murine EGF is replaced by Pro in an attempt to stabilize this turn conformation to produce a variant of mEGF with increased activity relative to that for the native protein. Ser25 is also replaced by Ala, which is found at position 25 in human EGF (hEGF), as a more conservative replacement. Receptor binding studies demonstrate that both mutations produce about a 30% reduction in binding affinity, which is shown to result from local changes within the loop or minor perturbations of residues neighboring the loop rather than from long-range perturbations of the-sheet of the N-terminal subdomain. The type I-turn appears to remain intact in both mutants; however, replacement with Pro seems to introduce more flexibility into this region of the protein. These results demonstrate that perturbation of this-turn has little effect on EGF-receptor interactions.Abbreviations EGF epidermal growth factor - h human - m murine - TGF type transforming growth factor - NMR nuclear magnetic resonance - [S2A]mEGF mEGF missing the N-terminal asparagine and with the serine at position 2 replaced by alanine - [S2A,S25A]mEGF and [S2A, S25P]mEGF replacement of serine at position 25 in [S2A]mEGF by alanine and proline, respectively - ¹²⁵I-mEGF ¹²⁵I-labeled mEGF - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum - HEPES N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - BSA bovine serum albumin - COSY correlated spectroscopy - DQCOSY double-quantum filtered COSY - NOESY nuclear Overhauser spectroscopy - NOE nuclear Overhauser effect - TOCSY total correlation spectroscopy - ³ J(H-H^N) vicinal spin-spin coupling constant between amide proton and -proton - DSS 2,2-dimethyl-2-silapentane-5-sulfonate - chemical shift in ppm - ppm parts per million,     

表皮生长因子对细胞核转录活性的直接作用

表皮生长因子（ＥＧＦ）对纯化的鼠胚成纤维细胞（Ｃ_３Ｈ_１０Ｔ_１／２Ｃ１８）胞核~３Ｈ－ＵＴＰ掺入有明显的促进作用。这一作用在ＥＧＦ与细胞核共同培养９０ｍｉｎ时达到高峰。该作用是ＥＧＦ浓度依赖性的。ＥＧＦ对β射线转化的上述细胞的纯化细胞核,亦同样有促进转录作用。本实验所用的细胞核经光镜,电子显微镜观察,细胞膜、细胞浆标志酶检测及放射性同位素示踪法鉴定,未检出有核外成份的污染。本实验结果为膜受体信号传递途径以外的ＥＧＦ对核直接作用途径的存在提供了证据。     

SESAME: A least-squares approach to the evaluation of protein structures computed from NMR data

Summary A method is proposed for defining a probability distribution on an ensemble of protein conformations from a 2D NOE spectrum, while at the same time back-calculating the experimental spectrum from the ensemble. This enables one to assess the relative quality and significance of the conformations, and to test the consistency of the ensemble as a whole with the experimental spectrum. The method eliminates the need to integrate the cross-peak intensities and is surprisingly insensitive to random noise in the spectrum. In this communication, these advantages are demonstrated by applying the method to simulated data, for which the correct result is already known.Abbreviations NOE nuclear Overhauser effect - BPTI basic pancreatic trypsin inhibitor - rmsd root-mean-square deviation     

Epidermal growth factor receptors destined for the nucleus are internalized via a clathrin-dependent pathway

The epidermal growth factor (EGF) transduces its actions via the EGF receptor (EGFR), which can traffic from the plasma membrane to either the cytoplasm or the nucleus. However, the mechanism by which EGFR reaches the nucleus is unclear. To investigate these questions, liver cells were analyzed by immunoblot of cell fractions, confocal immunofluorescence and real time confocal imaging. Cell fractionation studies showed that EGFR was detectable in the nucleus after EGF stimulation with a peak in nuclear receptor after 10 min. Movement of EGFR to the nucleus was confirmed by confocal immunofluorescence and labeled EGF moved with the receptor to the nucleus. Small interference RNA (siRNA) was used to knockdown clathrin in order to assess the first endocytic steps of EGFR nuclear translocation in liver cells. A mutant dynamin (dynamin K44A) was also used to determine the pathways for this traffic. Movement of labeled EGF or EGFR to the nucleus depended upon dynamin and clathrin. This identifies the pathway that mediates the first steps for EGFR nuclear translocation in liver cells.     

Identification of key residues involved in fibril formation by the conserved N-terminal region of Plasmodium falciparum merozoite surface protein 2 (MSP2)

Merozoite surface protein 2 (MSP2) from the human malaria parasite Plasmodium falciparum is expressed as a GPI-anchored protein on the merozoite surface. MSP2 is assumed to have a role in erythrocyte invasion and is a leading vaccine candidate. Recombinant MSP2 forms amyloid-like fibrils upon storage, as do peptides corresponding to sequences in the conserved N-terminal region, which constitutes the structural core of fibrils formed by full-length MSP2. We have investigated the roles of individual residues in fibril formation and local ordered structure in two peptides, a recombinant 25-residue peptide corresponding to the entire N-terminal domain of mature MSP2 and an 8-residue peptide from the central region of this domain (residues 8–15). Both peptides formed fibrils that were similar to amyloid-like fibrils formed by full-length MSP2. Phe11 and Ile12 have important roles both in stabilising local structure in these peptides and promoting fibril formation; the F11A and I12A mutants of MSP2_8–15 were essentially unstructured in solution and fibril formation at pH 7.4 and 4.7 was markedly retarded. The T10A mutant showed intermediate behaviour, having a less well defined structure than wild-type and slower fibril formation at pH 7.4. The mutation of Phe11 and Ile12 in MSP2_1–25 significantly retarded but did not abolish fibril formation, indicating that these residues also play a key role in fibril formation by the entire N-terminal conserved region. These mutations had little effect on the aggregation of full-length MSP2, however, suggesting that regions outside the conserved N-terminus have unanticipated importance for fibril formation in the full-length protein.     

Combined use of 13C chemical shift and 1Hα−13Cα heteronuclear NOE data in monitoring a protein NMR structure refinement

Summary A large portion of the ¹³C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28°C has been determined at natural isotope abundance. Sequence-specific ¹³C assignments are reported for 100% of the assignable C, 96% of the C, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C chemical shifts for regions of regular -sheet structure. These assignments also provide the basis for interpreting ¹H–¹³C heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these ¹H–¹³C HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated C values can be used to monitor the refinement of this protein's structure, particularly for -sheet regions. Improved agreement between calculated and observed values of C is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, C, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.Abbreviations HSQC heteronuclear single-quantum coherence spectroscopy - PFG pulsed-field gradient - TOCSY ¹H-¹H total correlation spectroscopy - EGF epidermal growth factor - mEGF murine EGF - hEGF human EGF - hTGF human type- transforming growth factor - DIPSI spm-locking pulse sequence - NOE nuclear Overhauser effect - HNOE heteronuclear Overhauser effect     

Structural study of copper(I)-bleomycin

Previous NMR studies on Cu(I)-bleomycin have suggested that this adduct has a geometry distinct from Fe(II)BLM. The coordination chemistry of this bleomycin derivative has been investigated through the extension of the NMR data reported previously, and the use of molecular dynamics calculations. The data collected from the NMR experiments support the coordination to the metal center of the primary and secondary amines in beta-aminoalanine and the pyrimidine ring. The detection in the NMR spectra of the signal derived from the amide hydrogen in beta-hydroxyhistidine indicates that this amide is protonated in Cu(I)-bleomycin, precluding participation of the pyrimidinyl carboxamide nitrogen in the coordination of Cu(I), as previously reported. Three-dimensional solution structures compatible with the NMR data have been assayed for Cu(I)-bleomycin for the first time by way of molecular dynamics calculations, and two models showing four and five coordination have been found to be those that better fit the experimental data. In both models the primary amine in beta-aminoalanine is coordinated such that it is located on the same side, with respect to the coordination cage, as the peptide linker fragment. This result seems important for the favored models to be compatible with either their possible oxidation to become one of the reported structures for Cu(II)BLM, or their transformation into Fe(II) adducts able to cause DNA damage.     

妊娠期糖尿病小鼠胎盘EGFR的表达与其发病关系的研究

目的:研究妊娠期糖尿病小鼠胎盘表皮生长因子受体(EGFR)的表达,探讨EGFR表达与妊娠期糖尿病发病的关系。方法:采用链脲佐菌素建立妊娠期糖尿病(gestational diabetes mellitus,GDM)小鼠模型,对照组为正常妊娠小鼠,腹腔注射等量缓冲溶液。测定母鼠体重、血糖;计算胎鼠的存活率;测定胎鼠、胎盘重量,计算胎盘效率;RT-PCR、免疫组化分别测定GDM组和对照组胎盘EGFR m RNA和EGFR蛋白的表达。Pearson相关性分析用于母鼠血糖与EGFR表达的相关性分析。结果:GDM组母鼠体重和血糖均高于对照组(P0.01);GDM组胎鼠、胎盘重量及胎盘效率均高于对照组(P0.01);RT-PCR和免疫组化结果显示GDM组胎盘EGFR m RNA和EGFR蛋白的表达与对照组相比差异有统计学意义(P0.01)。GDM组小鼠血糖值与其胎盘EGFR的表达具有相关性(r=0.582,P0.05)。结论:GDM导致胎盘EGFR表达升高,EGFR并不是GDM的发病因素,EGFR是预防GDM的潜在靶点。     

Acute Regulation of the Epidermal Growth Factor Receptor in Response to Nerve Growth Factor

PC12 cells possess specific receptors for both nerve growth factor and epidermal growth factor, and by an unknown mechanism, nerve growth factor is able to attenuate the propagation of a mitogenic response to epidermal growth factor. The differentiation response of PC12 cells to nerve growth factor, therefore, predominates over the proliferative response to epidermal growth factor. We have observed that the addition of nerve growth factor to PC12 cells rapidly produces a decrease in surface 125I-epidermal growth factor binding capacity. Unlike previously described nerve growth factor effects on 125I-epidermal growth factor binding capacity, which required several days of nerve growth factor exposure, the decreases we report occur within minutes of nerve growth factor addition: A 50% decrease in 125I-epidermal growth factor binding capacity is evident at 10 min. This rapid nerve growth factor response is concentration dependent; inhibition of 125I-epidermal growth factor binding is detectable at nerve growth factor levels as low as 0.2 ng/ml and is maximal at approximately 50 ng/ml, consistent with known ranges of biological activity. No demonstrable differences in the rate of epidermal growth factor receptor synthesis or degradation were observed in cells acutely exposed to nerve growth factor. Scatchard analysis revealed that acute nerve growth factor treatment decreased the number of both high- and low-affinity 125I-epidermal growth factor binding sites, while the receptor affinity remained unchanged. We have also investigated the involvement of various potential intracellular mediators of nerve growth factor action and of known intracellular modulatory systems of the epidermal growth factor receptor for their capacity to participate in this nerve growth factor activity.     

Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos)

The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-month-old alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis.     

Specific epidermal growth factor receptors on porcine thyroid cell membranes

The influence of islet-activating protein (IAP), a Bordetella pertussis toxin, was studied on adenylate cyclase and GTPase activities in rat adipocyte membranes. Pretreatment of rats or intact rat adipocytes with IAP did not affect adenylate cyclase inhibition by the stable GTP analog, GTP gamma S, whereas inhibition by GTP was abolished. Concomitantly, activation of the adipocyte enzyme by sodium and its inhibition by nicotinic acid were prevented. Furthermore, IAP treatment of adipocyte membranes prevented nicotinic acid-induced stimulation of a high affinity GTPase. The data suggest that a GTP-hydrolyzing system involved in the inhibitory regulation of adenylate cyclase is the target of IAP's action.     

阿霉素肾病大鼠表皮生长因子及其受体的表达

目的研究阿霉素肾病大鼠肾组织中表皮生长因子(EGF)及其受体EGFR的表达分布以及表达量与尿蛋白之间关系。方法选择第5天、14天、28天作为动态观察的时点,同期设立正常对照。采用荧光定量RT-PCR、免疫组织化学及计算机图像定量分析EGF mRNA以及EGF、EGFR蛋白在肾组织的表达,同时测定24 h尿蛋白定量。WT1和EGFR双重免疫组化确定EGFR在肾小球内确切细胞定位。结果阿霉素注射后第5天,EGFmRNA即较正常增高,28 d明显增高并高于5 d和14 d。正常对照组EGF阳性细胞主要分布于远曲小管和髓袢,阿霉素组EGF还在集合管和近曲小管上表达;EGF阳性表达范围和强度随尿蛋白增加而增加;EGFmRNA表达量以及EGF在肾小管中的表达强度与24 h尿蛋白量呈正相关。肾小管上皮细胞广泛表达EGFR,阿霉素组EGFR在小管表达均高于正常,但组间各时点差异无显著性;随尿蛋白增加EGFR在肾小球内表达逐渐增多。EGFR在肾小球和肾小管中的表达强度均与24 h尿蛋白量呈正相关。WT1和EGFR双重免疫组化显示阿霉素肾病组EGFR可在足突细胞上表达,正常组则无。结论阿霉素肾病大鼠的肾小球脏层上皮有EGFR的表达。EGF/EGFR可能参与了阿霉素肾病的发病过程以及蛋白尿的形成。     

Determination of melarsoprol in biological fluids by high-performance liquid chromatography and characterisation of two stereoisomers by nuclear magnetic resonance spectroscopy

The analysis of melarsoprol in whole blood, plasma, urine and cerebrospinal fluid is described. Extraction was made with a mixture of chloroform and acetonitrile followed by back-extraction into phosphoric acid. A reversed-phase liquid chromatography system with ultraviolet detection was used. The relative standard deviation was 1% at concentrations around 10 μmol/l and 3–6% at the lower limit of determination (9 nmol/l in plasma, 93 nmol/l in whole blood, 45 nmol/l in urine and 10 nmol/l in cerebrospinal fluid). Melarsoprol is not a stable compound and samples to be stored for longer periods of time should be kept at −70°C. Plasma samples can be stored at −20°C for upt to 2 months. Chromatography showed that melarsoprol contains two components. Using nuclear magnetic resonance spectroscopy the two components were shown to be diastereomers which slowly equilibrate by inversion of the configuration at the As atom.     

人神经干细胞的体外生物学特性

本实验利用有丝分裂因子,体外诱导生成人神 经干细胞(NSCs),观察其生长特性并进行鉴定。取胎龄10-22周的大脑半球,分散细胞后种于添加表皮生长因子(EGF,20ng/ml)和/或碱性成纤维生长因子(bFGF,20ng/ml)的培养基中。利用免疫组织化学方法鉴定分化后的细胞类型。同时,进行细胞克隆分析、传代培养及端粒酶活性检测。结果显示:NSCs呈悬浮生长的干细胞球,其特异性抗原nestin阳性。NSCs具有增殖能力,可连续传代而不丢失其增殖和多分化潜能的干细胞特性。撤除EGF和bFGF的作用,细胞停止分裂,并分化为神经元、星形胶质细胞和少突胶质细胞。克隆分析显示NSCs生长呈密度依赖性。人NSCs表达较低的端粒酶水平,并随培养时间延长而下调。研究表明,利用有丝分裂因子,可在体外成功诱导生成人NSCs,其生长,分化受内外源因素的调节,相关的机制还有待阐明。