Analysis of proteins in normal and regenerating rat liver using two-dimensional electrophoresis

The major proteins of homogenate, cytosol, nuclei and nuclear membrane extract from normal and regenerating rat liver were studied by two-dimensional electrophoresis with a view of detecting proteins involved in DNA replication regulation. Essential quantitative differences in three out of 200 polypeptides separated as spots and dyed with Coomassie R-250 on two-dimensional maps were revealed. The content of the p38 nuclear protein (Mr congruent to 38 kD, pI congruent to 4) increases 6-8-fold in the S-phase. The level of another nuclear protein, p50 (Mr congruent to 50 kD, pI congruent to 6.5) decreases 2-3-fold. The cytoplasmic protein p35 (Mr congruent to 35 kD, pI congruent to 8) also decreases 2-3-fold. Moreover, the p40 protein (Mr congruent to 40 kD, pI congruent to 6) whose content in the nuclei sharply rises up to 20 times after sham operation was revealed.     

长日照处理对牵牛开花抑制作用研究 Ⅱ.光周期处理与牵牛花芽分化及基因活动的关系

单个光周期暗期长度短于12h时,牵牛植株营养生长旺盛,开花受到抑制,并且出现了诱导光周期处理（ISD）子叶中没有的二种蛋白质或多肽（pI4.1,MW16.5kD;pI4.2,MW16．5kD）。连续光照处理（ICL）子叶内出现了短日照处理（ISD）子叶内没有的体外翻译蛋白质分子量为17．4kD的Poly（A~ ）mRNA。牵牛子叶内的这些变化可能与抑制牵牛花芽分化有一定的关系。     

热休克蛋白、蛋白水解酶和磷酸酶在大鼠肝再生中的含量和活性变化

本文以２／３肝切除（ｐａｒｔｉａｌ ｈｅｐａｔｅｃｔｏｍｙ,ＰＨ）大鼠为模型,探讨了ＰＨ后酸性和碱性磷酸酶（ａｃｉｄ ａｎｄ ａｌｋａｌｉｎｅ ｐｈｏｓｐｈａｔａｓｅｓ,ＡＣＰ和ＡＫＰ）,构成性热休克蛋白７０／诱导性热休克蛋白６８（ＨＳＣ７／ＨＳＰ６８）,酸性和中性蛋白水解酶在肝再生期间（０－１４４ｈ）的动态变化。结果显示,在肝切除后的肝再生期间;（１）ＡＣＰ和ＡＫＰ均出现两个活性高峰（４和４８ｈ     

Specific Proteins in Chloroplasts of Photoperiod-Sensitive Genic Male-Sterile Rice

The chloroplast proteins of a male-sterile mutant Nongken 58S, namely “Hubei Photoperiod-sensitive Genic Male-sterile Rice”, which is male sterile under long day (LD) cycles and fertile under short day (SD) cycles, and its original cultivar Nongken 58 (Oryza sativa L. subsp, japonica, Nongken 58) at seedling stage and photoperiod sensitive stage of fertility transition could be resolved into at least 20 major and more than 70 minor protein components on two-dimensional gel electrophoresis, with molecular weights ranging from 10kD to 67kD and isoelc points (pI) from 4.3 to 8.5. The mutant Nongken 58S had twospecific proteins with molecular weight of 45kD(pI6.7) and 61 kD (pI6.0) as compared with Nongken 58. Another 61 kD(pI6.2) protein was more in Nongken 58S than in Nongken 58. The existance of these proteins was not influenced by SD or LD treatment. The results showed difference in patterns of gene expression between Nongken 58S and Nongken 58. A possible function of these proteins in relation to regulation of sterility was discussed.     

小麦幼芽水分胁迫诱导蛋白的特征

水分胁迫（－1．2MPaPEG-6000）处理萌动的小麦种子,24h后诱导小麦幼芽产生41．5kD蛋白,其含量随着胁迫时间延长明显增加,48h时含量最高,到72h后不再变化。复水后,该蛋白消失;再胁迫48h时则又出现,其含量与处理24h时相当。41．5kD诱导蛋白主要位于细胞器膜上,细胞质中几乎不存在。41．5kD蛋白主要溶于10％NaCl提取液中,其等电点为pl5．65。该蛋白的氨基酸组成中,脯氨酸含量最高,其次为丙氨酸、天冬氨酸、谷氨酸、甘氨酸,没有发现半胱氨酸和组氨酸。     

乌龙岭’龙眼胚胎发育时期特异性蛋白质的变化

应用IEF-SDS-PAGE技术分析龙眼胚胎分化发育过程中蛋白质组分的变化。结果表明,在各发育阶段大多数蛋白质组分的电泳图谱基本一致,但也有变化。其中花后38d存在TE1(27．1kD、p,7．3),TE2(17．5kD、pI8．2)2个特异蛋白,45d存在TE3(11．4kD、pI7．6),TE4(13．2kD、pI9．9)2个特异蛋白,52d存在TE5(22．6kD、pI7．2),TE6(18．6kD、pI8．3),TE,(23．5kD、pI3．6)3个特异蛋白。31d胚胎电泳图谱中的蛋白质点数相对较多,表明此时蛋白质旺盛合成与积累,这与蛋白含量的变化基本一致。龙眼胚胎发育过程中特异蛋白的出现或消失．对胚胎的分化发育具有重要作用。     

Isolation of the mRNA encoding rat liver catechol-O-methyltransferase

A highly specific, well characterized rabbit antiserum to purified rat liver catechol-O-methyltransferase (COMT; EC 2.1.1.6) and the procedure of polysome immunoadsorption have been used to isolate a messenger RNA which encodes a single polypeptide when translated in vitro. Western blotting and immune fixation have shown multiple active forms of the enzyme to exist; a major, soluble one with MW of 23,000 and pI of 5.2 and another, membrane-bound one with MW of 26,000 and a pI of 6.2 (1). When translated in vitro, the purified message synthesizes a protein of molecular weight (MW) 23,000 and pI 5.2, values in agreement with those for purified enzyme reported by other investigators (2,3). Only the soluble form is seen after in vitro translation; the other immunoreactive proteins possibly arise due to post-translational modifications which do not occur in the lysate; or perhaps another mRNA exists. Cloning of the COMT cDNA will resolve this issue and should be feasible in light of our data indicating that the mRNA isolated here represents 0.46% of total rat liver polyadenylated message.     

Isolation and characteristics of surface proteins from Streptococcus group A

Cell wall surface proteins of group A Streptococcus type 29 were extracted with 1 M hydroxylamine pH 6.0. The purification procedure included fractionation with ammonium sulfate and gel filtration on Sephadex G-150. SDS polyacrylamide gel electrophoresis revealed a number of proteins (approximately 20) with molecular mass of 70 kD; the difference in Mr between the proteins was 5-10 kD. Isoelectrofocusing demonstrated that the proteins are either acid (pI = 3.7) or weakly alkaline (pI = 7.7). Possible reasons for the heterogeneity of Streptococcus cell wall surface proteins are discussed.     

应激心肌细胞蛋白质组双向凝胶电泳分析

采用双向凝胶电泳技术和计算机辅助的图像分析方法 ,对去甲肾上腺素诱导的应激心肌细胞与正常心肌细胞蛋白质进行分离和比较分析 .正常心肌细胞可分离 12 32± 5 6个蛋白点 ,蛋白点匹配率为 83 3%± 1 0 %.有 11种蛋白质在NE应激后发生了明显和稳定的质和量的改变 (P <0 0 5 ) ,其中 6种 (Mr pI :4 9 7kD 7 8,38 3kD 5 9,37 1kD 6 6 ,2 9 3kD 7 4 ,18 7kD 6 1,18 5kD 7 7)在应激后表达降低 ,4种 (Mr pI:4 7 6kD 5 5 ,31 9kD 4 4 ,2 6 6kD 4 6 ,33 2kD 8 1)在应激后表达增高 ,1种 (Mr pI:19 4kD 6 9)只在应激后发生表达 .这些差异表达的蛋白质可能参与了心血管应激反应乃至应激损伤发生的过程 .     

Expression of heat shock protein 27 in human renal cell carcinoma

Heat shock protein 27 (HSP27, Swiss-Prot accession number P04792) is a component of the large and heterogeneous group of chaperone proteins, and its main functions are inhibition of apoptosis and prevention of aggregation of actin intermediate filament. Modified expression of HSP27 has been described in several cancers including testis, breast, and ovaric cancer. In the present work, 18 renal cell carcinoma (RCC) tissues and homologous normal kidney tissues have been investigated for HSP27 expression by combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) separation and Western blotting immunodetection. The results showed significant differences either in expression and in HSP27 isoform numbers in RCC compared to normal kidney. The average number of isoforms was 21 in RCC and 15 in normal tissues with 4.5-5.9 pI range and 18-29 kDa M(r) range. The overexpression was also observed by immunohistochemistry on tissue sections. Only two of RCC samples showed less isoforms than homologous normal samples. Two isoforms were not detected using anti-Ser82 phosphorylated HSP27 antibody, neither in normal nor in RCC samples. Five of all the immunodetected isoforms were confirmed by mass spectrometry as HSP27, but no evidence of post-translational modifications was pointed out. The numerous isoforms observed in RCC are not consistent with data reported in the literature so far, and they might be due to different post-translational modifications such as phosphorylation and S-thiolation. Since activation of HSP27 seems to be involved in tumor proliferation and drug resistance, it would be crucial to correlate the severity of disease with the different isoforms from RCC samples to generate diagnostic and prognostic markers.     

Proteomic analysis of metabolic, cytoskeletal and stress response proteins in human heart failure

Human heart failure is a complex syndrome and a primary cause of morbidity and mortality in the world. However, the molecular pathways involved in the remodelling process are poorly understood. In this study, we performed exhaustive global proteomic surveys of cardiac ventricle isolated from failing and non-failing human hearts, and determined the regulatory pathway to uncover the mechanism underlying heart failure. Two-dimensional gel electrophoresis (2-DE) coupled with tandem mass spectrometry was used to identify differentially expressed proteins in specimens from failing (n = 9) and non-failing (n = 6) human hearts. A total of 25 proteins with at least 1.5-fold change in the failing heart were identified; 15 proteins were up-regulated and 10 proteins were down-regulated. The altered proteins belong to three broad functional categories: (i) metabolic [e.g. NADH dehydrogenase (ubiquinone), dihydrolipoamide dehydrogenase, and the cytochrome c oxidase subunit]; (ii) cytoskeletal (e.g. myosin light chain proteins, troponin I type 3 and transthyretin) and (iii) stress response (e.g. αB-crystallin, HSP27 and HSP20). The marked differences in the expression of selected proteins, including HSP27 and HSP20, were further confirmed by Western blot. Thus, we carried out full-scale screening of the protein changes in human heart failure and profiled proteins that may be critical in cardiac dysfunction for future mapping.     

Further evidence that phosphoprotein C23 (110 kD/pI 5.1) is the nucleolar silver staining protein

Previously we demonstrated a similar distribution between nucleolar organizing region-(NOR)-specific silver staining and localization of nucleolar phosphoprotein C23 (MW 110 kD/pI 5.1) [1, 2]. We now report that under fixation conditions which allow for antibody binding and subsequent silver staining, monoclonal antibody against protein C23 blocks NOR silver staining as well as silver staining in interphase nucleoli. Monoclonal antibody against nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1) did not block silver staining in either NORs or interphase nucleoli. These, along with earlier observations, provide evidence that nucleolar phosphoprotein C23 is the major silver staining protein of the nucleolus and that it is directly or indirectly associated with rDNA.     

红苋R104种子谷蛋白的电泳分析

通过单向水平变性聚丙烯酰胺凝胶等电聚焦（ＩＥＦ）、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（ＳＤＳ—ＰＡＧＥ）和双向电泳（ＩＥＦ×ＳＤＳ—ＰＡＧＥ）分析了红苋Ｒ１０４种子谷蛋白的亚基组成,亚基分子量和等电点分布。谷蛋白的双向电泳图谱可分辨出１００多个亚基成分．其主要亚基为：５４ｋＤ（ｐＩ７．１５）;３３ｋＤ（ｐＩ５．８２）;３１ｋＤ（ｐＩ６．９２;ｐＩ６．７０;ｐＩ６．６５）;２２ｋＤ（ｐＩ８．３４）;２０ｋＤ（ｐＩ６．９２;ｐＩ６５６）;１８ｋＤ（ｐＩ６．９２;ｐＩ７３５;ｐＩ７．７２;ｐＩ８．０５）。另外,还对ＩＥＦ方法进行了讨论。     

Proteomic analysis of altered protein expression in skeletal muscle of rats in a hypermetabolic state induced by burn sepsis

mRNA profiling has been extensively used to study muscle wasting. mRNA level changes may not reflect that of proteins, especially in catabolic muscle where there is decreased synthesis and increased degradation. As sepsis is often associated with burn injury, and burn superimposed by sepsis has been shown to result in significant loss of lean tissues, we characterized changes in the skeletal-muscle proteome of rats subjected to a cutaneous burn covering 20% of the total body surface area, followed 2 days later by sepsis induced by CLP (caecal ligation and puncture). EDL (extensor digitorum longus) muscles were dissected from Burn-CLP animals (n=4) and controls (sham-burned and sham-CLP-treated, n=4). Burn-CLP injury resulted in a rapid loss of EDL weight, increased ubiquitin-conjugated proteins and increased protein carbonyl groups. EDL protein profiles were obtained by two-dimensional gel electrophoresis using two immobilized pH gradient strips with overlapping pH range covering a pH 3-8 range. Seventeen spots were significantly altered in the Burn-CLP compared with the control group, representing 15 different proteins identified by peptide mass fingerprinting. The identities of three proteins including transferrin were further confirmed by liquid chromatography-tandem MS. The significant changes in transferrin and HSP27 (heat-shock protein 27) were verified by Western-blot analysis. HSP60, HSP27 and HSPbeta6 were down-regulated, along with HSP70, as detected by Western blotting. Six metabolic enzymes related to energy production were also down-regulated. A simultaneous decrease in chaperone proteins and metabolic enzymes could decrease protein synthesis. Furthermore, decreased HSPs could increase oxidative damage, thus accelerating protein degradation. Using cultured C2C12 myotubes, we showed that H2O2-induced protein degradation in vitro could be partially attenuated by prior heat-shock treatment, consistent with a protective role of HSP70 and/or other HSPs against proteolysis.     

Analysis and separation of synaptosomal membrane proteins

Synaptosomal membrane proteins solubilized with 8% CHAPS-8 M urea were analyzed with twodimensional electrophoresis (2DE). The membrane proteins were resolved up to 250 spots on a 2DE map, ranging in isoelectric points (pI) from 3.5 to 10.0 and molecular weights (MW) from 10 kDa to 200 kDa. Comparison of the mapped proteins of synaptosomal membranes with those of myelin and mitochondorial membranes revealed that synaptosomal membrane proteins were characteristic in the area of pI from 4.0 to 7.5 and MW from 20 kDa to 130 kDa, and that at least 30 spots were synaptosomal membrane-specific proteins. Most of these 30 proteins have not been previously described, named, and characterized Serial numbers (from SY1 to SY30) were assigned to the proteins on the map in order to investigate them systematically. A preliminary attempt to separate synaptosomal membrane proteins was carried out using a reversed-phase HPLC system. Several proteins could either be isolated or enriched. SY10 (pI 4.6; MW 56 kDa) was one of these proteins, and was of particular interest for its unusual behavior on the reversed-phase column, and for its binding to an immobilized protein A-gel.     

Analysis of individual CNS protein synthesis

A procedure for labeling rat CNS proteins in vivo which is useful for behavioral and pharmacological studies has been developed. Intraventricular administration of³⁵S-methionine through bilateral indwelling cannulae provided reproducible and highly specific radiolabeling of proteins from frontal cortex (FC), parietal cortex (PC), occipital cortex (OC), striatum (ST), septal nuclei (SN), amygdala (AM), hippocampus (HIP), thalamus (TH), brain stem (BS) and cerebellum (CB). Relative rates of synthesis of over 200 individual proteins were subsequently analyzed by 2DGE. Regional analysis demonstrated increased labeling of a protein of MW 28 kD and pI 6.4 in the hippocampus that was barely detectable in striatum of control rats. In heat-shocked animals, there was increased relative synthesis of the 74 kD Heat Shock Protein in both the septal nuclei and hippocampus.     

莲子贮存蛋白的主要亚基及积累模式

红莲（NelumbonuciferaGaertn）成熟于叶总蛋白含量达24．35g／100g干样品,其贮存蛋白（SP）的积累模式与豆科种子相似,即随成熟度的提高,SP猛增至占总蛋白含量的86％以上。对莲子不同发育阶段子叶蛋白SDS-PAGE图谱的光密度测定表明：莲子叶蛋白有12个主要SP亚基（SP1－12）。按分子量（MW）大小和积累顺序可分3组：A组是98kD和93kD的2个亚基,MW最大,积累最晚,但量最多;B组是3个亚基,MW为55－50kD,大小居中,积累最早,量最少;C组的7个亚基MW最小,在27－14kD之间,积累较早,量较多。对莲的不同品种、不同器官进行分析后发现,它们都有1个主峰为68kD的多连峰的代谢蛋白亚基,可能是莲共有的蛋白亚基。     

Isolation of a novel rat testicular metalloprotein binding cadmium and zinc

Various testicular metal-binding proteins having apparent mol wt in the range of 10–30 kD have been demonstrated by gel filtration of¹⁰⁹Cd- or⁶⁵Zn-labeled cytosol, but in no case has a purified metalloprotein been isolated that contains stoichiometric amounts of the metal. The purpose of this work was to purify from rat testes a testes-specific 30 kD Cd-binding protein (Cd-testin) following in vitro addition of¹⁰⁹Cd to testis cytosol. Conventional purification methods similar to those used for purification of metallothionein could not be used because Cd was not retained in stoichiometric amounts by the 30 kD species when these methods were employed. However, using ammonium sulfate fractionation, hydrophobic interaction and gel filtration chromatography, a 30 kD protein containing 2.6 mol of Cd/ mol of protein was isolated. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the isolated protein contained one major polypeptide with a mol mass of 22 kD and a pI of 4.6 (22 kD/pI 4.6) and two minor polypeptides (16 kD/pI 4.6 and 10±4 kD/pI 6.3) Two-dimensional gel electrophoresis demonstrated that the 22 kD species is a major low mol mass (<60 kD) protein in rat testic cytosol. The 22 kD protein was not detectable in cytosol of rooster testis, a tissue that is insensitive to Cd-induced damage and devoid of the 30 kD Cd-binding protein. Gel filtration and hydrophobic interaction chromatography of¹⁰⁹Cd- and⁶⁵Zn-labeled cytosol demonstrated that¹⁰⁹Cd and⁶⁵Zn cochromatography with the 30 kD protein. The function of this novel 30 kD testicular metal-binding protein is not known, but our work and other studies suggest that its occurrence in testes is linked to the production of a unique 22 kD polypeptide.     

肾综合征出血热纯化疫苗的SDS-PAGE分析

为了证明蛑综合征出血热纯化疫苗的主要成分坦病毒蛋白,采用出血热纯化疫苗经浓缩后进行SDS－PAGE和Western-blotting分析。结果 经SDS－PAGE显示,肾综合征出血热纯化疫苗有三条蛋白带,分子量分别约为70kD、55kD和50kD,与汉坦病毒三种结构蛋白（糖蛋白G1、G2和核蛋白NP）的分子量相符;经Western-blotting显示,分子量50kD的蛋白带反应阳性,分子量70kD和55kD的蛋白带无反应,认定出血热纯化疫苗的主要成分为汉坦病毒蛋白,主要由G1、G2和NP三种结构蛋白构成。