Actin and myosin during pollen germination

Actin and myosin from pollen tubes of Lilium davidii were studied by using immunoblotting, Dot_Blot and myosin Ca 2+_ATPase analysis. On immunoblotting of the total soluble pollen tube proteins, anti_α_actin antibody labelled a polypeptide approximately 43 ku, which is considered to be the actin of lily. The mRNA encoding actin in ungerminated pollen and germinated pollen were both undetectable in our experiments. A myosin exhibited Ca 2+_ATPase activity, with a native molecular weight of 460 ku has been identified by using immunoblotting. A polypeptide of about 205 ku and a polypeptide of about 20 ku were the heavy chain and a set of light chain of the myosin, which can crossreact with anti_skeletal muscle myosin heavy chain monoclonal antibody and anti_skeletal muscle myosin light chain (20 ku) monoclonal antibody, respectively. The Ca 2+_ATPase activities of myosin in crude extracts of germinated pollen were positively related to the growth rates of pollen tubes.     

Correlation between platelet gelsolin levels and different types of coronary heart disease

Gelsolin is an important cytoskeletal protein of platelets and studies have shown a close relationship between gelsolin and cardiovascular disease.However,the role of gelsolin in the development of coronary heart disease(CHD) is unclear.In this study,we record the distribution of gelsolin in human platelets and plasma and its association with different types of CHD.This study included 114 cases,with 33 stable angina pectoris(SAP) cases,81 acute coronary syndrome(ACS) cases—composed of 39 unstable angina pectoris(UAP) and 42 acute myocardial infarction(AMI) cases,and 31 healthy control participants.Gelsolin concentration in platelet rich plasma(PRP) and platelet poor plasma(PPP),actin filament(F-actin) and Gc-globulin of PPP were determined by enzyme-linked immunoadsorbent assay(ELISA).The fluorescence intensity of CD62p and cytoplasmic calcium([Ca2+] i) in human platelets measured by flow cytometry.We also used turbidimetry to detect the platelet aggregation rate(PAR).We analyzed the correlation between platelet gelsolin concentration and CD62p or plasma F-actin levels among each different patient group.Compared with the control group,the gelsolin level in PRP of UAP and AMI groups increased significantly(P<0.01),while the gelsolin level in PPP of all the three patient groups decreased markedly(P<0.01),and the CD62p,PAR,[Ca2+] i of platelets,F-actin and Gc-globulin of the UAP and AMI groups increased significantly(P<0.01).Compared with the SAP group,the gelsolin level in PRP,the PAR,[Ca2+] i of platelets and CD62p of other two groups increased significantly(P<0.01),F-actin of the AMI group increased markedly(P<0.01).Platelet cytoskeleton protein dynamics vary among the different types of CHD.Platelet gelsolin levels are markedly increased and accompanied by increased platelet activity,F-actin and [Ca2+] i of ACS patients,while gelsolin levels in PPP are markedly lower.Abnormally increased platelet gelsolin levels show high positive correlation with the level of platelet activity.Therefore,platelet gelsolin might be a novel molecular marker and/or a new potential therapeutic target of anti-platelet therapy of ACS.     

Polymerization of fluorescent analogue of plant actinin vitro andin vivo

Maize pollen actin has been labeled with Oregon Green 488 iodoacetamide. A yield of 3 mg fluorescent actin analogue has been obtained from 10 mg of maize pollen actin, which is 99% in purity and the dye/protein ratio is 72%. In the presence of Mg²⁺ and K⁺, the fluorescent actin analogue polymerized into filamentsin vitro. Green fluorescent filaments were observed when the fluorescent actin was introduced into living plant cells by microinjection, indicating that the fluorescent actin analogue functions similarly to the native actin.     

Characterization of an endo-1,4-β-glucanase from Lilium longiflorum that functioned in pollen germination and pollen tube growth

Endo-β-glucanases play vital roles in the regulation of pollen tube growth. Here, a previously identified endo-l,4-β-glucanase from Lilium Iongiflorum （lily）, named LlpCell, was expressed in Escherichia coli, purified, and further investigated for its physiological function. The recombinant LlpCell protein hydrolyzed carboxy-methylcellulose （CMC） and exhibited activity towards laminarin from Eisenia. arborea and 1,3：l,4-β-glucan of barley. The pH for the optimum activity was 6.0 and the value of Km calculated from CMC was 5.0 mg/mL. Adding EDTA resulted in the total loss of the enzymatic activity, and this effect could be restored by the addition of Ca^2＋. Western blotting analysis showed that LlpCell protein was present in pollen grains and rehydrated pollen grains, and the amount of the protein was increased during pollen germinating, but not in the pollen tube. Consistently, the immunofluorescence labeling study with the antibody against LIpCell also indicated the presence of LIpCell at the begin-ning of germination, but not in the elongating pollen tube. Furthermore, incubation of LlpCell with pollen at the beginning of pollen germination increased the germination percentage and the length of pollen tube. All of these results suggested that LlpCell could play an important role in the regulation of lily pollen germination and the initiation of pollen tube growth.     

Determination of Cd-MT in <Emphasis Type="Italic">Pteria penguin</Emphasis> with indirect non-competitive ELISA

Cadmium-metallothionein (Cd-MT) of Pteria penguin was used to immunize domestic rabbit in order to obtain polyclonal antibody against Cd-MT. Using the anti-Cd-MT antibody, a method of indirect non-competitive enzyme-linked immunosorbent assay (non-competitive ELISA) was established to detect shellfish Cd-MT. In this case, the minimum detectable concentration of P. penguin Cd-MT was (4.563±0.051) ng/mL. The linear range of detection was 9.75-2.0×104 ng/mL. Intra-assay relative standard deviation (RSD) was less than 11.6%, and inter-assay RSD less than 6.7%. The recovery rates ranged from 83.7% to 119.0%. The polyclonal antibody against Cd-MT of P. penguin can also be used to detect MT of other four types of shellfish. In the crude extracts from different organs of P. penguin, the MT concentrations determined by this method matched well with the concentrations of cadmium detected by the atomic absorption spectroscopy.     

Membrane skeleton spectrin in pollen and pollen tube

Immuno-western blots, immunofluorescence and immuno-gold labeling methods were used to study the existence and distribution of a homologue of the membrane skeleton spectrin in the pollen and pollen tube ofLilium davidii Duch. A spectrin homologue was found to exist in the pollen and pollen tube, distributed on membranes of secretory vesicles near Golgi apparatus.     

Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain

Gelsolin is representative of a class of actin-modulating proteins found in lower eukaryotes to mammals, which sever actin filaments. Gelsolin found in the cytoplasm of cells is functionally similar to a mammalian plasma protein of similar size, originally called ADF or brevin. Human plasma and rabbit macrophage gelsolins differ by the presence of a 25-amino-acid residue extension on plasma gelsolin which appears to account for the difference in relative molecular mass (Mr) between the proteins as assessed by SDS-polyacrylamide gel electrophoresis (PAGE), 93,000 (93K) and 90K, respectively. Here we report the isolation of full-length human plasma gelsolin complementary DNA clones from a HepG2 library. The inferred amino-acid sequence reveals the presence of a signal peptide, a long tandem repeat that matches the actin-binding domains of gelsolin, a tetrapeptide present in actin and extended regions of identical sequence with rabbit macrophage gelsolin. Southern blot analysis indicates that a single gene in the haploid genome encodes both protein forms.     

Palaeoenvironmental changes from pollen record in deep sea core PC-1 from northern Okinawa Trough, East China Sea during the past 24 ka

A pollen record of core PC-1 from the northern Okinawa Trough, East China Sea （ECS）, provides information on vegetation and climate changes since 24 cal. kaBP. A total of 103 samples were palynologically analyzed at 8 cm intervals with a time resolution of 230 a. Four pollen zones are recognized： zone Ⅰ （812-715 cm, 24.2-21.1 cah kaBP）, zone Ⅱ （715-451 cm, 21.1-15.2 cah kaBP）, zone Ⅲ （451-251 cm, 15.2-0.8 cah kaBP）, zone Ⅳ （251-0 cm, 10.8-0.3 cal. kaBP）, corresponding to Late MIS 3, Last Glacial Maximum （LGM）, deglaciation and Holocene, respectively. The LGM is characterized by the dominance of herbs, mainly Artemisia, and high pollen influx, implying an open vegetation on the ex- posed continental shelf and a cool and dry climate. The deglaciation is a climate warming stage with Pinus percentage increased and Artemisia percentage decreased and a rapid sea-level rise. The Holocene is characterized by predominance of tree pollen with rapid increase in Castanea-Castanopsis indicating the development of mixed evergreen and deciduous broad-leaved forest and a warm, humid climate. Low pollen influx during the Holocene probably implies submergence of the continental shelf and retreat of the pollen source area. The vegetation indicated by pollen assemblage found in this upper zone is consistent with the present vegetation found in Kyushu, Japan. Originating from the humid mountain area of North Luzon of the Philippines, Tasmania and New Zealand, Phyllocladus with sporadic occurrence throughout PC-1 core probably suggests the influence of Palaeo-Kuroshio Current or intense summer monsoon. The observed changes in Pinus and Herbs percentage indicate fluctuations of the sea level, and high Pinus percentage corresponds to high sea level. Spectrum analysis of the pollen percentage record reveals many millennial-scale periodicities, such as periodicities of 6.8, 3.8, 2.2, 1.6 ka.     

利用多克隆抗体检测纺锤体蛋白基因INMAP在肝癌细胞中的表达

 为探究纺锤体蛋白INMAP 的功能及其在细胞恶性增殖中发挥的作用,制备INMAP 多克隆抗体.利用0.1 mmol/L IPTG于16℃诱导His-INMAP 融合蛋白进行原核表达,SDS-PAGE 及免疫印迹检测蛋白表达.4.0 mol/L 尿素处理包涵体获得可溶性融合蛋白,镍亲和柱分离纯化融合蛋白抗原并检测蛋白浓度及纯度.以所获抗原免疫4 只Balb/c 小鼠,收集心脏抗血清.以纯化前、后的原核表达产物作为抗原,检测INMAP 多克隆抗体的特异性.通过免疫印迹分析INMAP 在正常肝细胞系L-02 及5个肝癌细胞系(PLC、HepG2、SUN449、SMMC-7721 和BEL-7402)中的表达差异.结果显示,His-INMAP 融合蛋白主要以包涵体形式存在,经4.0 mol/L 尿素处理可获得可溶性目的蛋白,且纯化后的抗原纯度较高(94.1%).INMAP 多克隆抗体能与INMAP 抗原特异结合.INMAP 在6 种肝细胞中具有多态性,除PLC 外,在其他肝癌细胞中其基因表达水平显著下调.本研究制备的INMAP 多克隆抗体特异性高,为INMAP 基因功能的进一步研究奠定了基础.     

Effects of ubiquitin-proteasome pathway on mouse sperm capacitation, acrosome reaction and in vitro fertilization

Chlortetracycline (CTC) fluorescence patterns were used to study changes in the patterns B and AR of mouse sperm after incubation with reagents that would block the UPP. They were the monoclonal antibody against ubiquitinated proteins—UCP1; the polyclonal antibody against ubiquitin—anti-Ub, and a special inhibitor against proteasome—ALLN. Furthermore, we treated the capacitated sperm or the eggs with these reagents separately and tested whether the normal in vitro fertilization was blocked or not. Results illustrate that UCP1, anti-Ub, and ALLN have little effects on sperm capacitation and acrosome reaction, but they do inhibit fusion of mouse sperm with eggs, which suggests that UPP play an important role in mouse in vitro fertilization.     

Effects of temperature on the expression of STN7 and phosphorylation of LHCII proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In Arabidopsis thaliana, STN7 kinase is required for phosphorylation of LHCII and for state transitions. In this paper, a hydrophilic polypeptide, derived from the amino acid sequence of STN7, was conjugated to a carrier protein, bovine serum albumin （BSA）, to obtain the polyclonal antibody. Immunogenicity and specificity of the polyclonal antibody were evaluated by agar gel immunodiffusion （AGID） test and Western blot analysis. The results show that besides the phosphorylation of LHCII proteins, also the expression of STN7 was regulated by temperature conditions. In addition, the change tendency of LHCII proteins phosphorylation was not only coherent with expression of STN7 with respect to increasing temperature, but also closely related to state transitions. These results would provide useful information for studying regulatory mechanism of LHCII proteins phosphorylation and expression of STN7.     

P-5m八肽多克隆抗体的制备及应用

目的制备P-5m八肽的多克隆抗体并进行初步鉴定和应用,为研究P-5m八肽的功能及作用机制获得重要的实验工具.方法将9-氟甲氧羰基(Fmoc-)固相合成法合成的P-5m八肽纯化后与载体蛋白-血蓝素联接;取偶联后的多肽皮下注射免疫新西兰白兔,并加强免疫得到抗血清,以C18反相色谱分离柱分离纯化;通过间接ELISA方法测定血清效价;利用Transwell实验和Western blot方法初步鉴定多克隆抗体拮抗P-5m八肽抑制肿瘤细胞转移的效果.结果纯化后的抗体经间接ELISA测定效价均大于1∶160 000,表明获得高效价的多抗;通过Transwell实验证明该抗体能够封闭P-5m对肿瘤细胞侵袭的抑制能力;通过Western blot方法证实此抗体效价较高,特异性较强;该多克隆抗体能够封闭P-5m在SW620细胞中上调E-cadherin表达的作用,并阻遏P-5m对MMP-2和MMP-9表达的下调作用,与对照组比较差异具有统计学意义(P0.05).结论 P-5m多克隆抗体可用于Transwell和Western blot等实验,为研究P-5m八肽的功能及抗肿瘤转移作用机制提供实验依据.     

整合素连接激酶相关磷酸酶（ILKAP）抗体的制备及其特异性分析

以整合素连接激酶相关磷酸酶（ILKAP）全蛋白为抗原, 采用腿部肌肉注射和耳缘静脉注射相结合的方法对新西兰大白兔进行免疫, 共免疫6次, 最
终获得ILKAP抗血清. 采用Protein A agrose柱纯化抗体, 获得ILKAP的多克隆抗体. 免疫印迹分析表明, 所获得的ILKAP多克隆抗体具有较高的特异性, 间接ELISA方法测定ILKAP多克隆抗体的滴度为1 ∶1 600. 免疫荧光分析表明, 所获得的ILKAP多克隆抗体在细胞水平应用效果较好.     

拟南芥雄性不育突变体opw(only pollen wall)候选突变基因的克隆

在T-DNA插入突变体Salk_059463株系的群体中,筛选到两株雄性不育突变体,对TDNA序列上的一对引物进行PCR鉴定,结果表明:其基因组中没有T-DNA插入.遗传分析表明这两株雄性不育突变体由同一单个隐性基因控制,引起不育的主要原因是从花药发育的第8期开始,小孢子细胞质内容物逐渐减少直至消失,到花药发育的第12期,药室内的小孢子只剩下一个花粉壁空壳,故该突变体命名为opw(only pollen wall).利用图位克隆的方法对OPW基因进行了定位,结果表明OPW基因位于第二条染色体上分子标记T28M21和T3G21之间的12 kb区间内,该区间内一共有21个基因注释.通过克隆区间内的基因并测序发现opw-1突变体基因组中At2g40140基因编码序列的外显子在第289和第290个碱基之间插入了一个A碱基,而opw-2突变体基因组中At2g40140基因编码序列的外显子在第412和第413个碱基之间插入了一个T碱基,造成的编码序列移码使第424至第426碱基成为终止密码子,故At2g40140是编码OPW的候选基因.     

兔抗PEB1多克隆抗体的制备及鉴定

摘要：【目的】利用纯化的空肠弯曲菌表面蛋白PEB1,制备其多克隆抗体。【方法】用PEB1蛋白作为抗原免疫兔,分离血清得到兔抗PEB1多克隆抗体,用ELISA法检测抗体效价,并用Western-Blotting验证。【结果】PEB1蛋白免疫兔后,得到了兔抗PEB1多克隆抗体,间接ELASA法检测表明兔抗PEB 1血清效价达到1：10 000,Western印记检测表明,该抗体能与PEB1蛋白发生特异性结合,其特异性与敏感性均较好。【结论】获得了高效价的PEB1多克隆抗体。     

Quantitative relationships between vegetation and several pollen taxa in surface soil from North China

According to the vegetation investigation and pollen analysis of surface samples sampled along a precipitation gradient of the Northeast China Transect (NECT), several pollen taxa, includingPinus, Betula, Quercus, Tilia, Acer, Ulmus, Artemisia, Chenopodiaceae, Gramineae and Cyperaceae, were chosen to make the regression and correlation analyses. The results indicated that there exists a close relationship between vegetation and pollen taxa in surface samples. The regression parameters for ten taxa in the forests in the eastern part of NECT were different from those in the steppes in the western part.Pinus, Betula, Artemisia and Chenopodiaceae, which have large slope and y-intercept terms, were over-representative taxa.Acer, Gramineae and Cyperaceae, which have small slope andy-intercept terms, were under-representative taxa.Quercus, Tilia andUlmus whose slope terms have negative correlation withy-intercept terms were equi-representative taxa. The pollen taxa with large slope or largey-intercept terms have small variability coefficients, implying that the slope andy-intercept terms for these pollen taxa are of high accuracy in the estimation of plant abundance from pollen frequencies.     

Identification and function analysis of spectrin-like protein in pollen tubes of lily （Lilium davidii Duch）

The elongation of pollen tube is an important process of sexual reproduction in higher plant. Cytoskeleton plays a major regulatory role in the elongation of pollen tubes. But whether membrane skeleton is involved in the pollen tube elongation is not clear. In this study, immunochemical detection of spectrin-like protein has been carried out in pollen tubes. By use of 2-dimensional electrophoresis(2DE) and western blotting, two spectrin-like proteins are found, one is 150 kD, and the other is 105 kD, with pl being 4.54 and 4.39, respectively. 150 kD spectrin-like protein is located in plasma membrane of pollen tube and 105 kD spectrin-like protein is located in cytoplasm, probably functioning as a subunit to form a dimmer (210 kD) in vivo. The elongation of pollen tubes is inhibited after spectrin antibody was injected into a growing pollen tube. These results suggest that spectrin-like proteins exist in pollen tube and play an important regulating role in the elongation process of pollen tubes from lily.