p53和Bcl-2蛋白在舌鳞癌组织中的表达及意义

目的 研究p53和Bcl-2蛋白在舌鳞癌和癌前病变中的表达及意义。方法 采用免疫组化SP法检测38例舌鳞癌（A组）、20例13腔癌前病变（B组）及10例正常口腔舌上皮组织（C组）中p53和Bcl-2蛋白的表达情况。结果比较A、B、C三组，p53和Bcl-2蛋白的表达在A组与C组、B组与C组之间，P均〈0．05，在A组与B组之间，P〉0．05；ps3蛋白异常表达在淋巴结转移组显著高于无转移组，P〈0．05；Bcl-2蛋白高表达与p53蛋白高表达之间无相关性，P〉0．05。结论p53和Bcl-2蛋白高表达与舌鳞癌的发生、发展密切相关。     

食管鳞癌中HIF-1α、Bax、Bcl-2的表达及意义

采用RT-PCR及免疫组化技术检测食管鳞癌和其正常食管上皮中低氧诱导因子（HIF-1α）的表达，用免疫组化SP法检测Bax和Bcl-2蛋白在食管鳞癌及其正常上皮中的表达。结果：食管鳞癌中HIF-1α基因和蛋白阳性表达率分别为96．0％和94．0％，正常食管上皮中未见表达，其阳性表达与食管鳞癌的组织学分级、pTNM分期及淋巴结转移呈正相关（P均〈0．05）。Bax和Bcl-2蛋白阳性表达率分别为80．0％和76．0％，正常食管上皮中未见表达。Bax蛋白表达水平与食管鳞癌淋巴结转移呈正相关（P〈0．05），Bcl-2蛋白在不同组织学分级间表达差异显著（P〈0．01）。HIF-1α蛋白与Bax、Bcl-2蛋白之间表达呈正相关（P〈0．05）。认为HIF-1α是食管鳞癌发生发展的一个关键基因，HIF-1α、Bax、Bcl-2在食管鳞癌发生发展过程中有协同作用，联合检测这三者在食管鳞癌中的表达有助于其早期诊断、治疗和预后判断。     

Id3、C—erbB-2、PCNA在乳腺癌中的表达及意义

目的研究分化／DNA结合抑制因子3（Id3）、人类表皮生长因子受体2（C—erbB-2）和增殖细胞核抗原（PCNA）在乳腺癌中的表达及其与乳腺癌的关系。方法采用免疫组化SABC法分别检测70例乳腺癌、40例乳腺癌癌旁组织、20例乳腺良性肿瘤、20例正常乳腺组织Id3、C—erbB2、PCNA蛋白的表达。结果乳腺癌Id3、C—erbB-2、PCNA的阳性表达率分别为88．6％、32．9％、95．7％,癌旁组织阳性表达率分别为35％、20％、67．5％,乳腺良性肿瘤阳性表达率分别为10％、5％、95％,正常乳腺组织阳性表达率分别为5％、0、40％。乳腺癌Id3、C—erbB-2、PCNA的表达增高。Id3、C．erbB-2、PCNA蛋白阳性表达率和表达强度与年龄、淋巴结转移、肿瘤大小、pTNM分期无相关性。C—erbB-2、PCNA与组织学分级有关。Id3与C—erbB-2呈正相关,C—erbB-2与PCNA呈负相关。结论乳腺癌Id3、PC—NA、C—erbB-2表达上调,联合检测三者对乳腺癌预后、治疗评估有重要意义。     

乳腺癌组织中nm23、PS2蛋白的表达及临床意义

目的探讨乳腺癌组织中的nm23蛋白、PS2蛋白表达及其临床意义。方法采用免疫组化SP法检测63例乳腺癌组织与56例乳腺良性疾病组织中的nm23蛋白、PS2蛋白,分析nm23蛋白与PS2蛋白表达与患者年龄、组织学分级、临床分期、腋窝淋巴结转移、ER表达及预后的关系。结果nm23蛋白在乳腺癌组织中表达阳性率为76．19％（48／63）,与患者年龄无相关性（P〉0．05）,与组织学分级、临床分期、淋巴结转移及ER相关（P均〈0．05）。PS2蛋白在乳腺癌组织中表达阳性率为66．67％（42／63）,与临床分期无相关性（P〉0．05）,与年龄、组织学分级、淋巴结转移及ER表达相关。nm23蛋白和PS2蛋白在乳腺良性疾病中的表达均高于在乳腺癌组织中的表达水平。nm23蛋白与PS2蛋白均阳性的乳腺癌患者预后明显优于均阴性的乳腺癌患者（P〈0．05）。结论nm23蛋白、PS2蛋白可以作为预测乳腺癌预后的指标。     

PLK1和PCNA在乳腺癌组织中的表达及临床意义

目的 探讨乳腺癌组织中PLK1、增殖细胞核抗原（PCNA）的表达及其临床意义。方法 应用免疫组织化学S-P法检测32例乳腺癌患者癌组织及16例乳腺良性增生患者乳腺增生组织中PLK1、PCNA的表达。结果 乳腺癌组织中PLK1、PCNA的阳性表达率分别为56、3％（18／32）、71．9％（23／32），PLK1表达与年龄、组织学类型、肿瘤大小、淋巴结状态、肿瘤分期及人类表皮生长因子受体-2（HER-2）的表达等因素无关（P均〉0．05），与肿瘤分化程度、雌激素受体（ER）、孕激素受体（PR）表达有关（P均〈0．05）。PCNA的表达与肿瘤分化程度、ER表达有关（P均〈0．05）；与年龄、组织学类型、肿瘤大小、淋巴结状态、PR及HER-2表达等因素无关（P均〉0．05）。乳腺良性增生组织中PLK1、PCNA表达均为阴性或弱表达。结论 PLK1在乳腺癌组织中表达具有一定肿瘤特异性，有望成为乳腺癌治疗的新靶点。     

三阴性乳腺癌组织中p53、Ki-67、EGFR的表达变化及意义

目的观察三阴性乳腺癌（TNBC）组织中p53、Ki-67、表皮生长因子受体（EGFR）表达变化,探讨其临床意义。方法采用免疫组化SP法检测137例TNBC组织中p53、Ki-67、EGFR的表达,分析其与TNBC临床病理特征及预后的关系。以非三阴性乳腺癌（N—TNBC）组织为对照。结果TNBC组织中p53、Ki-67、EGFR阳性表达率分别为55．7％、94．2％、65．0％,均明显高于N-TNBC的43．6％、90．7％、48．1％（P均〈0．05）;p53、Ki-67、EGFR阳性表达与TNBC大小、组织学分级、TNM分期、脉管内癌栓、淋巴结转移及预后有关（P均〈0．05）,与患者年龄和绝经状态无关（P〉0．05）。术后3a生存的TNBC患者,其癌组织中p53、Ki-67、EGFR阳性表达率为53．8％、94．1％、61．0％,术后生存5a者分别为51．8％、92．7％、56．9％,均高于术后生存3、5a的N-TNBC患者（P均〈0．05）。结论TNBC组织中p53、Ki-67、EGFR的表达均上调,其可作为TNBC的重要的预后指标,其中EGFR还可以作为靶向治疗的重要参考依据。     

胃黏膜病变中突变型p53、Mdm2和PCNA蛋白表达及与幽门螺杆菌感染的关系

目的探讨胃黏膜病变中突变型p53、Mdm2、增殖细胞核抗原（PCNA）蛋白的表达及其与幽门螺杆菌（Hp）感染的关系。方法应用免疫组化PV-9000法染色及Giemsa染色检测胃黏膜病变中突变型p53、Mdm2、PC-NA蛋白的表达和Hp感染情况。结果在慢性浅表性胃炎（csG）、慢性萎缩性胃炎伴肠上皮化生（CAG伴IM）、不典型增生（Bys）及胃癌（GC）中,突变型p53、Mdm2、PCNA阳性表达随病变发展而逐渐升高,突变型p53在CSG组高于GC组,GC组高于CAG伴IM组（P均〈0．05）。不同胃黏膜病变之间Mdm2比较均有统计学差异（P〈0．05）。在同一病变中Hp阳性者突变型p53、Mdm2、PCNA阳性表达一般高于Hp阴性者,突变型p53在CAG伴IM组Hp阳性与阴性者间有统计学差异（P〈0．05）,PCNA和Mdm2在Dys组Hp阳性与阴性者有统计学差异（P〈0．05）。GC组中突变型p53、Mdm2、PCNA蛋白表达无相关关系。结论突变型p53、Mdm2和PCNA蛋白的表达及Hp感染共同参与胃黏膜病变的发生、发展。     

膀胱癌组织中Bcl-2、PCNA、p53的表达变化及意义

目的观察膀胱癌组织中Bcl-2、PCNA、p53的表达变化，并探讨其临床意义。方法用流式细胞术检测3l例膀胱癌组织中的Bcl-2、PCNA、p53，并与18例正常膀胱黏膜组织作对照。PI染色法和TUNEL法分别检测上述标本的细胞周期及凋亡细胞。结果31例膀胱癌组织中，Bcl-2阳性表达率为58．1％，p53为67．7％，PCNA中强度阳性。正常膀胱黏膜组织Bcl-2阳性表达率为16．7％、p53不表达、PCNA弱阳性，两者相比，P均〈0．01。Bcl-2、PCNA、p53的表达与膀胱癌的临床分级或分期相关。膀胱癌细胞周期分布异常，19例出现DNA异倍体，s期细胞增加，但未发现明显的周期阻滞现象。膀胱癌细胞凋亡增加。结论膀胱癌组织中Bcl-2、PCNA、p53过度表达，可出现DNA异倍体，均提示预后不良。     

非小细胞肺癌组织中VEGF、p16及p53蛋白的表达及意义

目的探讨血管内皮细胞生长因子（VEGF）、p16蛋白及p53蛋白在非小细胞肺癌（NSCLC）发生、发展中的作用。方法应用免疫组化SP法检测82例非小细胞肺癌组织中VEGF、p16及p53表达。结果p53、p16及VEGF总阳性表达率分别为42．7％、45．1％和32．9％。有淋巴结转移者VEGF阳性率显著高于无转移者（P〈0．05）。肺鳞癌中p16与P53同时表达异常（p16阴性表达、p53阳性表达）者VEGF阳性率高于其它表型（P均〈0．05）。p16、p53的异常表达在有淋巴结转移者中分别为34．6％、50．9％,无淋巴结转移者中分别为66．7％、25．9％（P均〈0．05）。p16和p53的异常表达在Ⅲ期患者中的阳性率分别为51．7％和60．O％,在Ⅰ和Ⅱ期分别为58．％和24．4％（P均〈0．05）。结论NSCLC血管生成与p16和p55表达异常有关。     

非小细胞肺癌Bcl-2基因蛋白表达及其与细胞凋亡和增殖的关系

目的探讨非小细胞肺癌Bcl-2蛋白表达与细胞凋亡和增殖的关系。方法采用SAB免疫组化法检测43例非小细胞肺癌及20例癌旁正常肺组织石蜡包埋标本Bcl-2蛋白及增殖细胞核抗原（PCNA）表达；用TUNEL法检测细胞凋亡。结果肺癌组织Bcl-2蛋白的表达显著高于癌旁肺组织，P〈0．05；肺癌组织中凋亡指数和增殖指数均明显高于癌旁正常肺组织，P〈0．01，Bcl-2表达阳性的肺癌组织细胞凋亡指数明显低于Bcl-2表达阴性组，P〈0．05；高细胞凋亡、增殖指数提示肺癌预后不良，P〈0．05。结论肺癌存在Bcl-2表达上调，Bcl-2异常表达在肺癌发生过程中可能起重要作用。     

In vivo protein stabilization based on fragment complementation and a split GFP system

Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.     

Multimolecule test-tube simulations of protein unfolding and aggregation

Molecular dynamics simulations of protein folding or unfolding, unlike most in vitro experimental methods, are performed on a single molecule. The effects of neighboring molecules on the unfolding/folding pathway are largely ignored experimentally and simply not modeled computationally. Here, we present two all-atom, explicit solvent molecular dynamics simulations of 32 copies of the Engrailed homeodomain (EnHD), an ultrafast-folding and -unfolding protein for which the folding/unfolding pathway is well-characterized. These multimolecule simulations, in comparison with single-molecule simulations and experimental data, show that intermolecular interactions have little effect on the folding/unfolding pathway. EnHD unfolded by the same mechanism whether it was simulated in only water or also in the presence of other EnHD molecules. It populated the same native state, transition state, and folding intermediate in both simulation systems, and was in good agreement with experimental data available for each of the three states. Unfolding was slowed slightly by interactions with neighboring proteins, which were mostly hydrophobic in nature and ultimately caused the proteins to aggregate. Protein–water hydrogen bonds were also replaced with protein–protein hydrogen bonds, additionally contributing to aggregation. Despite the increase in protein–protein interactions, the protein aggregates formed in simulation did not do so at the total exclusion of water. These simulations support the use of single-molecule techniques to study protein unfolding and also provide insight into the types of interactions that occur as proteins aggregate at high temperature at an atomic level.     

蛋白质棕榈酰化修饰与心血管疾病

蛋白质棕榈酰化修饰是一种广泛存在于生物体内的蛋白质翻译后脂质修饰。在大多数情况下,棕榈酸主要通过不稳定的硫酯键共价结合到蛋白质底物特定的半胱氨酸残基上,从而发生S-棕榈酰化修饰。由于硫酯键在一定的条件下会水解,引起蛋白质底物发生去棕榈酰化修饰,因此S-棕榈酰化修饰通常是可逆的。S-棕榈酰化修饰也与许多心血管疾病的发生和发展密切相关。本文综述了蛋白质棕榈酰化修饰和去棕榈酰化修饰过程,以及蛋白质棕榈酰化修饰与心律失常、动脉粥样硬化、肺动脉高压、心力衰竭和血栓性疾病等心血管疾病之间的关系,为心血管疾病的治疗提供新的方向。     

uPA、P-选择素蛋白在乳腺癌中的表达及意义

目的 探讨uPA与P-选择素蛋白在乳腺癌浸润、转移中的作用以及二者的关系。方法　采用免疫组化SP法检测uPA与P-选择素蛋白在乳腺纤维腺瘤、乳腺导管内癌及浸润性导管癌组织中的表达。结果 随着乳腺组织的恶性转化、浸润及淋巴结转移,uPA、P-选择素蛋白均呈高表达趋势(P<0.05),二者之间有明显的相关性(r=0.547,P<0.01)。结论 uPA与P-选择素蛋白参与了乳腺癌的浸润及淋巴结转移,联合检测二者,对乳腺癌的预后判断有一定的参考价值。     

Molecular in situ topology of Aczonin/Piccolo and associated proteins at the mammalian neurotransmitter release site

The protein machinery of neurotransmitter exocytosis requires efficient orchestration in space and time, for speed and precision of neurotransmission and also for synaptic ontogeny and plasticity. However, its spatial organization in situ is virtually unknown. Aczonin/Piccolo is a putative organizer protein of mammalian active zones. We determined by immunogold electron microscopy (EM) (i) the spatial arrangement (i.e., topology) of 11 segments of the Aczonin polypeptide in situ, and correlated it to (ii) the positioning of Aczonin-interacting domains of Bassoon, CAST/ELKS, Munc13, and RIM and (iii) the ultrastructurally defined presynaptic macromolecular aggregates known as dense projections and synaptic ribbons. At conventional synapses, Aczonin assumes a compact molecular topology within a layer 35 to 80 nm parallel to the plasma membrane (PM), with a "trunk" sitting on the dense projection top and a C-terminal "arm" extending down toward the PM and sideward to the dense projection periphery. At ribbon synapses, Aczonin occupies the whole ribbon area. Bassoon colocalizes with Aczonin at conventional synapses but not at ribbon synapses. At both conventional and ribbon synapses, CAST, Munc13, and RIM are segregated from Aczonin, closer to the PM, and Aczonin is positioned such that it may control the access of neurotransmitter vesicles to the fusion site.     

Structural relationships among proteins with different global topologies and their implications for function annotation strategies

It has become increasingly apparent that geometric relationships often exist between regions of two proteins that have quite different global topologies or folds. In this article, we examine whether such relationships can be used to infer a functional connection between the two proteins in question. We find, by considering a number of examples involving metal and cation binding, sugar binding, and aromatic group binding, that geometrically similar protein fragments can share related functions, even if they have been classified as belonging to different folds and topologies. Thus, the use of classifications inevitably limits the number of functional inferences that can be obtained from the comparative analysis of protein structures. In contrast, the development of interactive computational tools that recognize the “continuous” nature of protein structure/function space, by increasing the number of potentially meaningful relationships that are considered, may offer a dramatic enhancement in the ability to extract information from protein structure databases. We introduce the MarkUs server, that embodies this strategy and that is designed for a user interested in developing and validating specific functional hypotheses.     

From the Cover: In-cell thermodynamics and a new role for protein surfaces

There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge–charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.Classic theories about the effects of complex environments consider only hard-core repulsions (volume exclusion) and so predict entropy-driven protein stabilization (1–3). Here, we use the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) as a model to test this idea in living cells. SH3 exists in a dynamic equilibrium between the folded state and the unfolded ensemble (4). This two-state behavior (5) is ideal for NMR-based studies of folding. Fluorine labeling (6) of its sole tryptophan leads to only two ¹⁹F resonances (7): one from the folded state, the other from the unfolded ensemble (Fig. 1A). The area under each resonance is proportional to its population, ρ_f and ρ_u, respectively. These populations are used to quantify protein stability via the modified standard state free energy of unfolding,ΔGU,T°′=−RTlnρUρF,[1]where R is the gas constant and T is the absolute temperature. Furthermore, the width at half height of each resonance is proportional to the transverse relaxation rate, which is an approximate measure of intermolecular interactions (8–10). Thus, this simple system yields both quantitative thermodynamic knowledge and information about interactions involving the folded state and the unfolded ensemble.Open in a separate windowFig. 1.Fluorine spectra acquired at 298 K, in buffer (A) and cells (B). The blue trace is from the postexperiment supernatant and shows that the red spectrum arises from protein inside cells. Stability curves (C) in buffer (black), in cells (red and green), and in 100 g/L urea (magenta). In-cell metabolite correction and analysis of uncertainties are discussed in Results and Discussion and Materials and Methods, respectively. Shaded regions are 95% confidence intervals. Error bars for buffer are smaller than the labels and represent the SD of three trials. Error bars for the in-cell data at 273, 298, and 313 K represent the SD of three trials. Stability in buffer (black) and solutions of 100 g/L BSA (blue) and lysozyme (red) at different pH values (D–F). The curve for buffer from C is reproduced in D. The net charges on SH3, BSA, and lysozyme (based on sequence) are shown. Error bars (298 K) represent the SD from three trials. Appearance of new resonances in the pH 3 BSA sample prevented extraction of thermodynamic parameters.To assess the enthalpic (ΔHU°′) and entropic (ΔSU°′) components, we measured the temperature dependence of ΔGU°′. These data were fitted to the integrated Gibbs–Helmholtz equation (11), assuming a constant heat capacity of unfolding, ΔCp,U°:ΔGU,T°′=ΔHU,Tref°′−TΔSU,Tref°′+ΔCp,U°′[T−Tref−T⁡lnTTref],[2]where T_ref is either the melting temperature, T_m (where ρ_f = ρ_u), or the temperature of maximum stability, T_s (where ΔSU°′ = 0) (11).     

Characterization of prolactin- and growth hormone-binding proteins in milk and their diversity among species

The present study was undertaken to identify and characterize the diversity and species distribution of soluble prolactin binding-protein (PRL-BP) and growth hormone-binding protein (PRL-BP) in mammalian milk. We previously divided mammalian serum GH-BP into four main groups and identified a GH-BP with shared lactogenic/somatogenic properties in rabbit, horse, dog, pig and cat (Type III species). Here we describe PRL-BP in milk of Type III species and show it is relatively conserved within the group, having similar characteristics in terms of binding affinity for hGH (0.74−5.5×10¹⁰ M⁻¹), specificity towards the lactogenic hormones and molecular weight (35 kDa), except for the more heterogeneous pig milk (43 to 88 kDa) Furthermore, high affinity PRL-BP was also demonstrated in sheep milk, having pure lactogenic specificity and an M_r of 35 kDa. Human milk contained a high affinity PRL-BP/GH-BP, which was recognized by both hPRL and hGH and also having an M_r of 35 kDa. In rabbit milk a separate GH-BP was also detected; it was clearly distinguished from the corresponding milk PRL-BP on the basis of its M_r of 44 kDa (vs. 32 kDa for PRL-BP), its shared lactogenic/somatogenic hormonal specificity (vs. purely lactogenic for PRL-BP) and also on the basis of its relative resistance to heating at 56°C for up to 3 h, while PRL-BP activity was completely destroyed within 30 min. This diversity of milk PRL-BP and GH-BP among mammalian species fits in with our earlier classification of serum GH-BP and also with the reported evolutionary rates of PRL and GH; this suggests these BPs may play important species-specific roles in the suckling newborn and/or maternal mammary gland, in keeping with the functions described for GH-BP.     

Physicochemical principles that regulate the competition between functional and dysfunctional association of proteins

To maintain protein homeostasis, a variety of quality control mechanisms, such as the unfolded protein response and the heat shock response, enable proteins to fold and to assemble into functional complexes while avoiding the formation of aberrant and potentially harmful aggregates. We show here that a complementary contribution to the regulation of the interactions between proteins is provided by the physicochemical properties of their amino acid sequences. The results of a systematic analysis of the protein–protein complexes in the Protein Data Bank (PDB) show that interface regions are more prone to aggregate than other surface regions, indicating that many of the interactions that promote the formation of functional complexes, including hydrophobic and electrostatic forces, can potentially also cause abnormal intermolecular association. We also show, however, that aggregation-prone interfaces are prevented from triggering uncontrolled assembly by being stabilized into their functional conformations by disulfide bonds and salt bridges. These results indicate that functional and dysfunctional association of proteins are promoted by similar forces but also that they are closely regulated by the presence of specific interactions that stabilize native states.