基于AFP+ AFP-L3+PIVKA-Ⅱ的电泳荧光仪性能分析

随机选择南充市某三甲医院门诊体检患者,参照美国临床实验室标准化协会（CLSI）EP-6A2文件,结合甲胎蛋白（AFP）及其异质体比率（AFP-L3）与异常凝血酶原（PIVKA-Ⅱ）试剂分析验证了全自动电泳荧光免疫分析仪μTASWakoi30的精密度、线性范围、准确性性能.结果表明,μTASWakoi30精密度CV均在10%以下,完全满足标准要求;AFP及PIVKA-Ⅱ在高值样本不同比例下通过实测均值与预测值回归拟合均线性良好;在不同校准品比和不同质量浓度水平下,回收率均在要求范围80%~120%内,准确性高.     

Microplate chemiluminescence enzyme immunoassay for the quantitative evaluation of carbohydrate antigen 72-4 in human serum

A highly sensitive and specific microplate chemiluminescence enzyme immunoassay （CLEIA） was developed for the quantitative evaluation of carbohydrate antigen 72-4 （CA72-4） in human serum, using luminol-H2O2 catalyzed by horseradish peroxidase （HRP） as the chemiluminescence system. The simple and quick determination was accomplished through a sandwich reaction mode. Several physicochemical parameters of the immunoreaction, including incubation conditions, antibody coating conditions, dilution ratio of anti-CA72-4-HRP conjugate, and chemiluminescence reaction time, were studied and optimized. The proposed method exhibited a linear range of 0-200 U/mL with correlation coefficient and detection limit of 0.9995 and 0.18 U/mL, respectively. The inter-assay and intra-assay coefficients of variation （CV） were both less than 10%. The average recovery of two clinical sera with low and high concentration CA72-4 was 99.3% and 98.7%, respectively. Normal tumor markers, including AFP, CEA, CA24-2, CA19-9 and CA15-3, did not cross-react with each other. The method＇s stability was evaluated by assessing its analytical performance after storing the immunoreagents at 4℃ and 37℃ for 7 days. Little difference was found, indicating satisfactory stability of the method. The present method has been successfully applied to the detection of CA72-4 human serum, and showed a good correlation with the commercially available ELISA kit （r^2=0.9383）. This method showed great potential in the fabrication of diagnostic kit for CA72-4, and could be well used in diagnosis of cancer in clinical practice.     

修饰合成冬鲽抗冻肽基因的设计和克隆

动物界和植物界密码子使用频率不同,冬Ｄｉｅ抗冻肽中丙氨酸占６０％而且偏爱ＧＣＣ,３８个Ａｌａ密友子中２３个为ＧＣＣ。我们设计合成抗冻肽基因时采用了植物基因常用密码子,用甘薯信号肽序列取了抗冻肽的ｐｒｅ序列,省略了Ｐｒｏ序列,并 信号肽下游二、四、八拷贝正向串联的成太的修饰基因,经测序证实与设计完成一致。利用ＱＩＡ表达系统,在大肠杆菌中实现了ＤＨＦＲ－成熟抗冻肽单体和二体的融合表达。     

肿瘤标志物联合检测在原发性肝细胞癌早期诊断中的应用

用生物发光免疫分析法、 比色法联合检测确诊的原发性肝细胞癌（53例）、 乙型病毒 性肝炎（80例）、 慢性乙肝合并肝硬化（34例）患者同份血清的肿瘤标志物甲种胎儿蛋白( AFP)和肿瘤特异性生长因子(TSGF)．　结果显示, 原发性肝细胞癌组血清TSGF和AFP与其他 组比较均有显著差异（P<0.01）; 原发性肝癌组中TSGF与AFP联合阳性检出率为96.2 %． 关键词: 生物发光免疫分析技术; 甲种胎儿蛋白; 肿瘤特异性生长因子; 原发性肝细胞癌     

Induction-dependent neural marker expression and electrophysiological characteristics of bone marrow mesenchymal stem cells that naturally express high levels of nestin

Under certain experimental conditions, bone marrow mesenchymal stem cells (MSCs) express neuronal phenotypes and neuronal markers, which suggests that they could be used to treat various neurological diseases. In the present study, MSCs were isolated from adult rat bone marrow, cultivated, and evaluated for neurotrophin expression profiles, as well as the potential to differentiate into functional neuronal-like cells in vitro. MSCs from passage 5 were pre-induced with DMEM/F12 medium containing 10% fetal bovine serum (FBS) and 10 ng/mL bFGF (fibroblast growth factor-2). Subsequently, a chemical inductor containing Dimethyl Sulphoxide (DMSO), Butylated Hydroxyanisole (BHA) and forskolin were used to induce neural expression of MSCs. Expression patterns of nestin, NF-200, and GFAP at time points before and after induction were detected by immunofluorescence. Nerve Growth Factor (NGF), brain-derived neurotrophic factor (BDNF) expressions in MSCs were evaluated by RT-PCR. The whole-cell patch clamp technique was utilized to elucidate the electrical behavior of MSC before and after 24-h differentiation induction. Immunofluorescence analysis revealed that MSCs expressed nestin (57.1% ± 6.9%), but not NF-200 or GFAP. Following neural induction, the cells exhibited a neuronal-like appearance. Nestin and NF-200 expression was positive in the neuronal-like cells, but GFAP expression was negative. After 6-, 12- and 24-h induction, the ratio of nestin-positive cells was 96.5% ± 1.9%, 88.1% ± 5.4%, and 33.5% ± 5.4%. NF-200 positive cells were 90.1% ± 2.9%, 97.5% ± 1.3%, and 98.1% ± 1.6%, respectively. However, prior to induction, MSCs already expressed NGF and BDNF. With a stimulus impulse of 40 mV, the density of the transient outward K current was (9.95 ± 4.85) pA/pF (n = 9) and (328.50 ± 30.62) pA/pF (n = 9) before and after induction, and the density of transient calcium ion currents was (−0.059 ± 0.027) pA/pF (n = 7) and (−6.66 ± 0.50) pA/pF (n = 7), respectively. Transient outward potassium currents and calcium ions currents gradually increased following induction. In addition, MSCs isolated from bone marrow exhibited characteristics of neuronal progenitor cells and expressed neurotrophins. These cells exhibited the capacity to differentiate into functional neuronal-like cells in vitro. These results suggested that MSCs express high levels of nestin and could be utilized for therapeutic strategies to treat nervous system diseases.     

New paleomagnetic results from the Yangzhuang Formation of the Jixian System,North China,and tectonic implications

A paleomagnetic study was carried out on the Yangzhuang Formation （-1350 Ma） of the Jixian System in the North China Block （NCB）. Detailed stepwise thermal demagnetization isolated two components. The soft component （component A） was interpreted as a remagnetization in the recent geomagnetic field. The hard component （component B） with higher unblocking-temperatures was carried by hematite. Its site-mean direction is D/I = 77.6°/-24.3°κ= 5.4, α 95 = 18.3°, N = 15 sites） before, and D/I = 72.2°/11.5° （ κ=24.6, α95 = 7.9°） after tilt correction. It passes a fold test（Mesozoic folding） at 99% confidence level and reversal test at 95% confidence level. The corresponding pole locates at 17.3°N, 214.5°E （dp = 4.1°, dm= 8.0°）. A best fit of paleomagnetic poles from the NCB, Baltica, Siberia and the apparent polar wander path for Laurentia suggests a long-lived connection between these paleo-continents between -1800and 1350 Ma.     

胡萝卜抗冻蛋白在大肠杆菌中的高效表达

以pET—11a为载体构建了胡萝卜抗冻蛋白非融合表达的重组质粒，并在大肠杆菌菌株BL21(DE3，pLysS)中成功地进行了高效表达。表达的蛋白质以包涵体的形式存在，相对分子质量为36800。经正交试验得到了优化的诱导表达条件。包涵体经各种缓冲液洗涤纯化、溶解、复性和超滤浓缩后，经SDS—PAGE电泳检测，纯度达单一带水平。生物学活性研究表明，重组表达的胡萝卜抗冻蛋白具有很强的热滞活性和提高细菌耐寒性的作用。     

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.     

Characterization of a FeMo cofactor-deficient MoFe protein from a nifE-deleted strain （DJ35） of Azotobacter vinelandii

A MoFe protein （△nifE Av1） with a purity of ～80% was purified from a nifE-deleted mutant of Azotobacter vinelandii DJ35. Compared with MoFe protein purified from wild-type strain OP （OP Av1）, △nifE Av1 had the same subunits composition, and had immune reaction with antibody to OP Av1, but its relative mobility in anaerobic native polyacrylamide gel electrophoresis （PAGE） was a little larger than that of OP Av1. Metal analysis showed that Mo and Fe contents of △nifE Av1 both apparently decreased. When complemented with OP Fe protein, △nifE Av1 had no C2H2-reduction activity, but it could be in vitro activated by FeMoco extracted from OP Av1. The circular dichroism （CD） spectrum of △nifE Av1 at ～450 nm was similar to that of OP Av1, while the EPR signal at g≈3.7 was absolutely silent, and the signal intensities at g≈4.3 and 2.0 decreased by 75% and 50%, respectively. The results indicated that △nifE Av1 purified from DJ35 was a FeMoco-deficient but P-cluster-containing MoFe protein.     

Cretaceous synfolding remagnetization components revealing tectonic rotation of the middle Yangtze fold belt

To reveal the deformation process of the middle Yangtze fold belt, we conducted a paleomagnetic study on Middle Triassic limestones and Middle to Late Jurassic sandstones from Wanzhou, Chongqing. Stepwise thermal and alternating field demagnetization were used to isolate the multi-component re-manent magnetizations. The Jurassic samples were overprinted by recent geomagnetic field, while three magnetization components were isolated from the Middle Triassic samples. A low temperature component (LTC) was isolated at temperatures below 200℃, an intermediate temperature component (ITC) at 200―360℃ and a high temperature component (HTC) at 400―460℃. The LTC is distributed around the present-day Earth magnetic field, probably a viscous component. Stepwise unfolding indi-cates that the maximum precision parameters of ITC and HTC components are achieved at 33±8% and 50±27% (with 95% confidence) unfolding, respectively. The best-clustered ITC mean direction, Dec = 11.2°, Inc = 45.2° (α₉₅ = 4.5°, N = 34), corresponding to a paleopole at 79.3°N, 219.5°E (d_p = 3.6°, d_m = 5.7°), is consistent with the Cretaceous reference direction of the South China Block (SCB). The best-clustered HTC mean direction (taking 70% unfolding), Dec = 24.2°, Inc =49.0° (α₉₅ = 3.6°, N = 23), corresponding to a paleopole at 69.2°N, 195.5°E (d_p =3.1°, d_m = 4.8°), suggests a clockwise rotation of 12.8°±3.5°. These synfolding remagnetization components clearly reveal that a clockwise rotation happened at the middle stage of folding, thus supporting that at least part of the variation in fold axis strikes is due to orocline rotation. Combined with published data, our analysis indicates that the Wan-zhou-Xiangxi segment of the middle Yangtze fold belt experienced oroclinal bending. Furthermore, a published post-folding component isolated from the Middle Triassic Puqi Formation suggests a 27.5°±5.8° clockwise rotation, confirming that at least 50% of the observed clockwise rotations in the eastern middle Yangtze fold belt can be attributed to oroclinal bending. The remagnetization data and geological evidence observed in the middle Yangtze fold belt suggest that collision between SCB and North China Block (NCB) probably lasted till the early period of Early Cretaceous.     

Gene mapping of a new coat mutation in mouse

Gene mapping of a mouse coat mutation has been investigated. First, 100 10-bp random primers were used to amplify DNA, but the mutation could not be located by this method because there were no correlation between the amplified products and coat phenotypes. Second, by usingIdh1, Car2, Mup1, Pgb1, Hbb, Es10, Es1, Mod1, Gdc1, Ce2, Es3 as genetic markers, linkage test crosses (two-point test) consisting of intercrossing uncovered BALB/c mice (homozygotes) to CBA/N and C57BV6 mice with normal hair and backcrossing the heterozygotes of the F1 to the uncovered BALB/c mice were made. It was soon evident that the mutation was linked toEs3 on chromosome 11. Furthermore, three-point test was made by usingEs3 and D11Mit8 (a microsatellite DNA) as genetic markers. The result showed that the mutation was linked toEs3 with the percentage recombination of (7.89 ± 2.19)%, and linked to Dl1Mit8 with the percentage recombination of (26.30± 3.57)%. The percentage recombination betweenEs3 and D11Mit8 was (32.90±3.81)%. The mutation was named Uncovered, with the symbolUncv. According to the recombinations, the loci order was D11Mit8-26.30±3.57-Uncv- 7.89 -2.19-Es3. From the location on the chromosome, it was concluded that the mutation was a new mutation which affected the skin and hair structure of mouse. TheUncv has entered MGD (Mouse Genome Database).     

Biological aspects on the cultures of the entomophthoralean fungus<Emphasis Type="Italic">Pandora delphacis</Emphasis> grown on broomcorn millets

Among entomopathogenic fungi the Entomophthor- ales plays an important role in natural control of insect pests through induction of epizootics in insect popula-tions[1—3]. Since most species of the fungal group are 4 - characteristic with their special capability of actively dis-charging conidia to favor infection and dissemination in host populations, they are ideal targets for epizootiologi-cal study, being considered as important microbial control agents. Currently, 66 species …     

Ovicidal activity of two fungal pathogens （Hyphomycetes）against Tetranychus cinnabarinus （Acarina：Tetranichidae）

The carmine spider mite, Tetranychus cinna-barinus, is an economically important pest that devastates varieties of crops worldwide and develops significant resistance to common chemical pesticides, most of which lack ovicidal activity. In the present study, two isolates of entomopathogenic fungi, Beuaveria bassiana SG8702 and Pae-cilomyces fumosoroseus Pfrl53, were bioassayed against T. cinnabarinus eggs at 25 ℃ under a photophase of 12 : 12 (L:D). Infected eggs on Vicia faba var. minor leaves failed to hatch due to distortion and shrinkage and had fungal outgrowths when maintained under moist conditions. Sprays of B. bassiana conidia to T. cinnabarinus eggs (on leaves) at the concentrations of 58, 298 and 1306 conidia/mm2 (3 replicates per concentration and 35-65 fresh mite eggs per replicate) resulted in corrected egg mortalities of 20.4±4.2%, 36.0±7.6% and 64.6±12.5% (F=43.14, P <0.01), respectively; sprays of P. fumosoroseus at 129, 402 and 2328 conidia/mm2 caused egg mortalities of 16.1±11.1%,     

A new technique for synthesis of 2,4-dichloro-thieno[3,2-d]pyrimidine

3-Amino-2-methoxycarbonylthiophene used as the main raw material,an intermediate(thiofuran siamese) 2,4-dichloro-thieno[3,2-d]pyrimidine was synthesized.The prod-uct was characterized by nuclear magnetic resonance(NMR).The first step yield was 84%(190 ℃) and the second step yield was 81.4%(105 ℃,m(intermediate):m(phosphorus oxychloride) = 1:6.4).The raw product was refined with a unique refining process,which can enhance the purity up to 99.5% to meet the requirement of condi-tions for the development and research of thiofuran siamese as a new medicine of anticarcinogen.Contrasted with the traditional technique,this process featured with cheap raw materials,possible for solvent recovery and reuse,low comprehensive cost,and suit-able for production on certain scale.     

Beta-helix structure and ice-binding properties of a hyperactive antifreeze protein from an insect

Insect antifreeze proteins (AFP) are considerably more active at inhibiting ice crystal growth than AFP from fish or plants. Several insect AFPs, also known as thermal hysteresis proteins, have been cloned and expressed. Their maximum activity is 3-4 times that of fish AFPs and they are 10-100 times more effective at micromolar concentrations. Here we report the solution structure of spruce budworm (Choristoneura fumiferana) AFP and characterize its ice-binding properties. The 9-kDa AFP is a beta-helix with a triangular cross-section and rectangular sides that form stacked parallel beta-sheets; a fold which is distinct from the three known fish AFP structures. The ice-binding side contains 9 of the 14 surface-accessible threonines organized in a regular array of TXT motifs that match the ice lattice on both prism and basal planes. In support of this model, ice crystal morphology and ice-etching experiments are consistent with AFP binding to both of these planes and thus may explain the greater activity of the spruce budworm antifreeze.     

大鼠肝细胞系的建立及其特性研究

从 Wistar 新生大鼠肝组织已成功获得正常肝细胞体外长期培养细胞系 NRL-84.细胞生长情况、分裂指数、染色体分析、软琼脂集落形成试验、相差显微镜及扫描电镜等多项指标观察.表明符合正常肝细胞生物学特征;用间接免疫荧光、对流免疫电泳、核酸原位杂交等检测甲胎蛋白,符合正常肝细胞的变动规律.正常新生大鼠的建立是国内首次报导,它将为进一步研究恶性肿瘤的发生发展机制提供了一个良好的实验模型和手段.     

Malvastrum yellow vein virus,a new Begomovirus species associated with satellite DNA molecule

Virus isolate Y47 was obtained from Malvastrum coromandelianum showing yellow vein symptom in Honghe, Yunnan Province. The complete nudcotide sequence of DNA-A was determined, it contains 2731 nuclcotides,having typical genomic organiTation of a begomovirns, encoding 6ORFs with 2ORFs [AVI(CP) and AV2] in virionsense DNA and 40RFs (ACl-AC4) in complementary-sense DNA. Comparisons show that the total DNA-A of Y47 has the highest sequence identity (77%) with that of Okra yellow vein mosaic virus-[201] (AJ002451), while less than 76% identities are found when compared with other begomoviruses. The molecular data show that virus isolate Y47 is a distinct begomovirns species, for which the name Maivastrum yellow vein vorus is proposed. Satellite DNA molecule (Y47β) was found to be associated with Y47 using the primers (beta01 and beta02) specific for DNAβ Y47β consists of 1348 nuclcotides, with a functional ORF (CI) in complemen-tary-sense DNA.Y47β has 62%--67% sequence identity with DNAβ molecule associated with Cotton leaf curl Muitan virus or Cotton leaf curl Rajasthan virus, while lower than 46% sequence identities are found when compared with other reported DNA[~ molecules. Relationship dendrograms show that DNAβ molecules are co-evolved with their help begomoviruses.     

Comparison of infection of different cell lines by uropathogenic Escherichia coli

Studying the interaction between uropathogenic Escherichia coil （UPEC） and uroepithelial cells is important in elucidating the pathogenesis of urinary tract infection. In this study, the African green monkey kidney cells （Vero）, human kidney carcinoma cells （Ketr-3） and bladder carcinoma cells （EJ） were infected by UPEC132, a clinical strain isolated from Tianjin, China, and were compared for their capacities to allow the adherence and invasion by this strain. The results revealed that all these cell lines could be attached and invaded by UPEC132. The adherence rates for Vero, Ketr-3 and EJ cells were （49,20 ±7.55）%, （55.22 ±4.09）% and （73.20 ±5.26）%, respectively, and invasion frequencies were （2.61 ±0.32）×10^-3, （3.00 ±0.34）×10^-3 and （3.25 ± 0.20）×10^-3, respectively. The statistical analysis showed that the adherence rate for EJ cells was significantly higher than those for the other two cell lines （P〈0.05）, and the invasion frequencies for EJ and Ketr-3 cells had no statistical differences （P〉0.05） but were higher than that for Vero cells （P〈0.05）. Three cell lines were detected for the receptors for P pill of UPEC by using indirect immunofluorescence. The results showed that receptors existed on the surfaces of all cell lines, and the highest distribution was found on the surface of EJ cells. Additionally, the invasion of EJ cells by recombinant UPEC132/pSELECT-GFP could be directly visualized using confocal microscopy. These data strongly implicated that EJ cells could be more easily infected by UPEC132 than the other cells, and thus could serve as a good experimental target for further investigation of UPEC infection.     

Preparation of high selectivity silicalite-1 membranes by two-step <Emphasis Type="Italic">in situ</Emphasis> hydrothermal synthesis

High selectivity silicalite-1 membranes were synthesized on silica tubes by in situ hydrothermal synthesis. Using a two-step synthesis, a membrane with a separation factor of 99 was prepared for separating an ethanol/water mixture at 60°C. The average (n = 4) flux and separation factor of the membranes were 0.47 kg m-2 h-1 and 89, respectively. The membranes exhibited high reproducibility, and the relative standard deviation of the average (n = 4) flux and separation factor were only 5.3% and 9.2%, respectively. These results suggest that silica is a suitable support for synthesis of high-performance silicalite-1 membranes.     

SHCSP与AFP检测肝癌研究

本文用SHCSP与AFP检测原发性肝癌126例,转移性肝癌6例,非肝癌患者141例,SHCSP法诊断敏感性90.48％,特异性90.07％,诊断效率90.26％,阳漏本9.52％,假阳性9.93％,LR(+)=9.11,LR(-)=0.11。而AFP法诊断敏感性70.63％,特异位88.65％,诊断效率80.15％,阳漏率29.37％,假阳性11.35％,LR(+)=6.22,LR(-)=0.33,检测SHCSP诊断肝癌比AFP优,且有非常显著性差别(P<0. 01),两法在诊断肝癌和排除肝癌有互补性,应联合检测,以提高肝癌诊断率。