Extended applications of electric cell-substrate impedance sensing for assessment of the structure-function of α2β1 integrin

Electric cell-substrate impedance sensing (ECIS) was applied to assess the structure-function of α2β1 integrin, receptor for collagen and laminin. On collagen-coated gold electrodes, expression of this integrin on human rhabdomyosarcoma (RD) cells (RDX2C2) yielded a five-fold increase in resistance when compared with mock transfected RD (RDpF) cells (34.5±5.2 versus 6.5±0.8 Ω/cell). An intermediate level of 16±2 Ω/cell was measured upon expression of an α2β1 mutant that lacked the α2 cytoplasmic domain (RDX2CO). On laminin, the resistance measured for RDX2C2 cells was also higher but only twice that of RDpF cells at 71±4 and 37±4 Ω/cell, respectively. In comparison, RDX2CO cells (38±4 Ω/cell), exhibiting no enhanced adhesive function, yielded a similar result to that of RDpF cells. On fibronectin, RDX2C2 and RDpF cells, exhibiting comparable levels of adhesion, were similar in resistance measurements at 85±5and 89±7 Ω/cell, respectively. It has been shown that deletion of α2 cytoplasmic domain results in dysregulated recruitment of the α2β1 mutant to focal adhesion complexes that mediate binding of fibronectin. RDX2CO cells on fibronectin, exhibiting reduced adhesive function, was associated with noticeably lower resistance (60±4 Ω/cell). Monitoring electroporation of the RD plasma membrane also indirectly validated cell attachment as reflected by the resistance measured. Results from this study demonstrated the potential of ECIS for study of the structure-function of βl integrin adhesion receptors.     

Imaging fiber microarray fluorescent ion sensors based on bulk optode microspheres

Optical imaging fibers with micrometer-sized wells were used as a sensing platform for the development of microarray optical ion sensors based on selective bulk extraction principles established earlier for optodes. Uniform 10 μm sized microspheres based on plasticized poly(vinyl chloride) containing various combinations of ionophores, fluoroionophores and lipophilic ion-exchangers were prepared for the detection of sodium, potassium, calcium and chloride, and deposited onto the wells of etched fiber bundles. Specifically, sodium sensing particles were based on tert-butylcalix[4]arene tetraacetic acid tetraethylester, potassium particles on 2-dodecyl-2-methyl-1,3-propanediyl bis[N-[5′-nitro(benzo-15-crown-5)-4′-yl]carbamate] (BME-44), calcium particles on an acrylic derivative of ETH 129 (AU-1) covalently attached to a methacrylic polymer, and chloride particles based on the anticrown ionophore [9]mercuracarborand-3 (MC-3). The fluorescence emission characteristics of individual microspheres were observed from the backside of the fibers and were found to selectively and rapidly change as a function of the sample composition. The optical characteristics of the particles were found to be comparable to that of corresponding thin optode films and particles deposited onto microscope glass slides. The measuring ranges (logarithmic molar concentrations) at pH 7.0 were found as −3 to 0 for sodium, −3.5 to −0.5 for potassium, −7 to −2 for calcium, and −5 to 0.5 for chloride. Selectivities were determined over other common electrolytes and found to be sufficient for physiological applications. The simultaneous deposition of sodium and chloride sensing particles was successfully performed, demonstrating that such microarray sensors are capable of simultaneously sensing multiple analytes. This technology is compatible with other microsphere-based fluorescent sensing principles, forming a promising total analysis platform for a variety of applications.     

Catalytic DNA-and RNA-hydrolyzing antibodies from milk of healthy human mothers

Various catalytically active antibodies (Abs), or abzymes, have been detected recently in the sera of patients with autoimmune pathologies, in whom their presence is probably associated with autoimmunization. Normal humans are generally not considered to have abzymes, since no obvious immunizing factors are present. Here is shown by different methods that IgG from the milk of normal females possesses both DNase and RNase activities. The activities were also present in the IgG F(ab′)₂ and Fab fragments. Affinity modification of IgG by the chemically reactive derivative of an oligonucleotide led to preferential modification of the L chain of IgG. After separation of the subunits by sodium dodecyl sulfate electrophoresis in a gel containing DNA, an in-gel assay showed DNase activity in the L chain. The L chain separated by affinity chromatography on DNA-cellulose was catalytically active. These findings speak in favor of the generation of cat alytic Abs by the immune system of healthy mothers. It is known that the treatment of adults with DNases and RNases offers protection from viral and bacterial diseases. Since breast milk protects the infants from infec tions until the immune system is developed, this raises the possibility that catalytic Abs like nucleases, may possess a protective role.     

Direct targeting of human plasma for matrix-assisted laser desorption/ionization and analysis of plasma proteins by time of flight-mass spectrometry

A method to analyze human plasma proteins without fractionation, directly applying a plasma-matrix mixture on the target plate of a matrix-assisted laser desorption/ionization-time of flight-mass spectrometer (MALDI-TOF-MS), has been described. Peaks of ionized plasma proteins could not be detected applying a mixture of an undiluted plasma sample and a matrix solution, but they appeared when the plasma was diluted before mixing with the matrix. Tenfold diluted plasma provided well-resolved protein peaks in the m/z range from 4000 to 30,000. The addition of a simple post-crystallization washing procedure performed on the target plate further improved the quality of mass spectra. We numbered 58 peaks in the range of 4-160 kDa and 32 out of which were assigned to the plasma protein species which have been reported. Especially high sensitivity and resolution were obtained in the region < 30 kDa, where multiple isoforms of apolipoprotein A-I, apolipoprotein A-II, apolipoprotein C-I, apolipoprotein C-II, apolipoprotein C-III, and transthyretin could be assigned. Various post-translational modifications are involved in the isoforms, e.g., proteolytic cleavage, glycosylation and chemical modifications. This method will become complementary with the present electrophoretic techniques, especially for the analysis of low-molecular-mass proteins.     

人类钙营养状况,缺钙后果及其对策

人类缺钙是世界性普遍存在的问题，中国人由于膳食结构缺陷及传统饮食习惯而使食物中钙的吸收受到影响，因而缺钙尤其普遍。     

Fluorescence immunological determination of immunoglobulin G in human serum by high-performance liquid gel-permeation chromatography

Summary A high-performance liquid gel-permeation chromatographic method is described for the determination of human serum immunoglobulin G (IgG) by separating the fluorescent immuno complex from the free fluorescence-labeled antibody. Fluorescence-labeled antibody used in this study was fluorescein isothiocyanate (FITC)-labeled Fab fragment goat anti-human IgG (anti-IgG Fab). Immuno complexes and antibody of different molecular sizes can be separated. FITC-labeled anti-IgG Fab was added to the serum and the mixture is passed through the column. An immuno complex separates as well-delineated peak in the column void volume, and was measured by the fluorescence of the column eluate (Ex=490nm, Em=520nm). The total analysis time for a serum sample was approximately 15min. The minimum detection limit was 25 mg/dl. The relative standard deviation was below 2% (peak area). The results of the HPL-GPC analysis correlate well with those obtained by laser nephelometric assay (r=0.992).     

抗人绒毛膜促性腺激素（HCG）单克隆抗体的研制和特性分析

以HCG为抗原,免疫BALB/c小鼠,取其脾细胞与小鼠Sp2/0—Ag14骨髓瘤细胞融合,经反复筛选和再克隆化,获得8株能稳定分泌抗HCG特异单克隆抗体的杂交瘤细胞株。对其中6个高效价单抗进行了特异性和抗原决定簇检测,选出两个抗相距较远不同决定簇的单抗(2D_4,4F_4)作进一步特性分析。结果显示:免疫球蛋白亚类鉴定,2D_4为IgGl亚类,4F_4为IgG2a亚类;亲和力常数(K_(affi)),2D_4为5.41±0.94×10~(-10)mol/L,4F_4为1.37±0.33×10~(-9)mol/L。证实该两种单克隆抗体可用于研制有关临床诊断试剂盒。     

离子交换法纯化重组类人胶原蛋白II的研究

用阳离子交换树脂CM-52分离纯化类人胶原蛋白II(HumanCollagen-likeBioproteinII,HCBII)。通过实验,分别确定了静态吸附和柱层析吸附的操作条件,即静态吸附:在pH4.0、NaCl0.15mol/L、进料浓度7g/L,处理量17.26ml/g树脂的条件下进行吸附,吸附时间为60min;柱层析:在pH4.0、NaCl0.15mol/L、进料浓度5g/L、流速5ml/min的条件下进行吸附。然后上柱,在pH4.0、NaCl0.30mol/L下进行脱附。实验表明,采用静态吸附的方式吸附HCBII,然后上柱洗脱,HCBII的吸附量可达到48.6mg/g树脂,回收率83.8%,操作时间短,分辨率高,最终纯化的HCBII达电泳纯,分子量为97kD。     

Application of laser ablation–inductively coupled plasma-mass spectrometry (LA–ICP–MS) to investigate trace metal spatial distributions in human tooth enamel and dentine growth layers and pulp

Human tooth enamel provides a nearly permanent and chronological record of an individuals nutritional status and anthropogenic trace metal exposure during development; it might thus provide an excellent bio archive. We investigated the micro-spatial distribution of trace metals (Cu, Fe, Mg, Sr, Pb, and Zn) in 196×339 m² raster pattern areas (6.6×10⁴ m²) in a deciduous tooth using laser ablation-inductively coupled plasma-mass spectrometry (LA–ICP–MS). Ablated areas include prenatal and postnatal enamel, the neonatal line, the dentine–enamel junction (DEJ), dentine, and the dentine–pulp junction. Topographic variations in the surface elemental distribution of lead, zinc, strontium, and iron intensities in a deciduous tooth revealed heterogeneous distribution within and among regions. ⁴³Ca normalized elemental intensities showed the following order: Sr>Mg>>Zn>Pb>Fe>Cu. Elevated zinc and lead levels were present in the dental pulp region and at the neonatal line. This study demonstrates the ability of LA–ICP–MS to provide unique elemental distribution information in micro spatial areas of dental hard tissues. Elemental distribution plots could be useful in decoding nutrition and pollution information embedded in their bio apatite structure.Presented in part at the 2002 Winter Conference on Plasma Spectrochemistry, Scottsdale, AZ, January 6–12, 2002. The poster was selected as an outstanding poster presentation.     

Non-aqueous capillary zone electrophoresis method for the analysis of paroxetine, tamoxifen, and their main metabolites in urine

The potential of non-aqueous capillary electrophoresis (NACE) was investigated for the simultaneous separation of paroxetine, tamoxifen, and their main metabolites. Baseline separation of the studied solutes was obtained on a  μm capillary using a non-aqueous buffer composed of 18 mM ammonium acetate and 1.1% acetic acid in 80:20 (v/v) methanol-acetonitrile, with a temperature and voltage of 22 °C and 15 kV, respectively. Clomipramine was used as internal standard. Aspects such as stability of the solutions, linearity, accuracy, precision and ruggedness were examined in order to validate the proposed method. Detection limits obtained for all the studied compounds ranged between 3.0 and 7.1 μg l⁻¹. The developed method is sensitive and robust and was used to determine paroxetine, tamoxifen, and their metabolites at clinically relevant levels in human urine. Before NACE determination, the samples were purified and enriched by means of an extraction-pre-concentration step with a pre-conditioned C₁₈ cartridge. Determination of these analytes in the urine of four females urines was demonstrated.