用三步法纯化重组人促红细胞生成素

在生物反应器中培养中国仓鼠卵巢细胞—促红细胞生成素(CHO-EPO)C2细胞株,培养上清液中重组人促红细胞生成素(rHuEPO)表达水平达2000～3000U/ml。培养上清经过三步纯化:第一步为反相柱色谱,可将样品浓缩约96.7％,其收集液经充分透析后进行第二步的DEAE-离子交换色谱,最后进行分子筛色谱,总回收率为30％以上,经纯化的rHuEPO比活性为1.5×10~8U/g蛋白,SDSPAGE一条带,扫描测试纯度达98％以上。     

Detection of Salmonella spp. in food by a rapid PCR-hybridization procedure.

A rapid and sensitive PCR-hybridization procedure for detection of Salmonella serovars in food samples was developed. This method is based on three subsequent steps: (1) extraction of nucleic acids from a 2 ml aliquot of the pre-enrichment medium used for the conventional culture method after 6 h of incubation at 37 degrees C; (2) amplification with primers selected from the sequences of invE and invA genes; (3) Southern blot and hybridization with a biotin labeled oligonucleotide probe. The entire procedure requires 30 h. The PCR-hybridization assay was able to detect as little as 50 fg of purified chromosomal DNA of S. typhimurium and 0.2 cfu g-1 of an artificially contaminated food sample. Of 245 food samples analyzed by culture and PCR-hybridization, 20 were positive by both methods and 16 were positive by PCR-hybridization only. None of the 209 PCR-negative samples tested positive by culture. The sensitivity, specificity, alpha and beta error values of the results of the PCR-hybridization procedure, compared with those of culture, were 100, 92.9, 0 and 7.1%, respectively. These results indicate that a short pre-enrichment and PCR-hybridization could be used as a screening test for the detection of Salmonella in food samples.     

Time variable osmotic pressures produced by coupled reactions as a possible cause of cell division

It has been shown in a previous paper that coupled reactions may produce diffusion phenomena which are characterized by highly asymmetric distributions of concentrations in a spherical cell. Under certain conditions such diffusion fields are unstable, the asymmetries in concentrations tending to increase indefinitely. This results in an increase of asymmetrically distributed osmotic pressures which may eventually result in a division of a cell. Constriction without elongation, as in cleavage, is studied. The process of division is brought about in this case by ordinary osmotic pressure, and not by the diffusion drag forces.     

Effect sizes in experimental pain produced by gender, genetic variants and sensitization procedures

Background

Various effects on pain have been reported with respect to their statistical significance, but a standardized measure of effect size has been rarely added. Such a measure would ease comparison of the magnitude of the effects across studies, for example the effect of gender on heat pain with the effect of a genetic variant on pressure pain.

Methodology/Principal Findings

Effect sizes on pain thresholds to stimuli consisting of heat, cold, blunt pressure, punctuate pressure and electrical current, administered to 125 subjects, were analyzed for 29 common variants in eight human genes reportedly modulating pain, gender and sensitization procedures using capsaicin or menthol. The genotype explained 0–5.9% of the total interindividual variance in pain thresholds to various stimuli and produced mainly small effects (Cohen''s d 0–1.8). The largest effect had the TRPA1 rs13255063T/rs11988795G haplotype explaining >5% of the variance in electrical pain thresholds and conferring lower pain sensitivity to homozygous carriers. Gender produced larger effect sizes than most variant alleles (1–14.8% explained variance, Cohen''s d 0.2–0.8), with higher pain sensitivity in women than in men. Sensitization by capsaicin or menthol explained up to 63% of the total variance (4.7–62.8%) and produced largest effects according to Cohen''s d (0.4–2.6), especially heat sensitization by capsaicin (Cohen''s d = 2.6).

Conclusions

Sensitization, gender and genetic variants produce effects on pain in the mentioned order of effect sizes. The present report may provide a basis for comparative discussions of factors influencing pain.     

Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure.

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.     

Cotton bracts are adapted to a microenvironment of concentrated CO2 produced by rapid fruit respiration

Background and Aims

Elucidation of the mechanisms by which plants adapt to elevated CO₂ is needed; however, most studies of the mechanisms investigated the response of plants adapted to current atmospheric CO₂. The rapid respiration rate of cotton (Gossypium hirsutum) fruits (bolls) produces a concentrated CO₂ microenvironment around the bolls and bracts. It has been observed that the intercellular CO₂ concentration of a whole fruit (bract and boll) ranges from 500 to 1300 µmol mol⁻¹ depending on the irradiance, even in ambient air. Arguably, this CO₂ microenvironment has existed for at least 1·1 million years since the appearance of tetraploid cotton. Therefore, it was hypothesized that the mechanisms by which cotton bracts have adapted to elevated CO₂ will indicate how plants will adapt to future increased atmospheric CO₂ concentration. Specifically, it is hypothesized that with elevated CO₂ the capacity to regenerate ribulose-1,5-bisphosphate (RuBP) will increase relative to RuBP carboxylation.

Methods

To test this hypothesis, the morphological and physiological traits of bracts and leaves of cotton were measured, including stomatal density, gas exchange and protein contents.

Key results

Compared with leaves, bracts showed significantly lower stomatal conductance which resulted in a significantly higher water use efficiency. Both gas exchange and protein content showed a significantly greater RuBP regeneration/RuBP carboxylation capacity ratio (J_max/V_cmax) in bracts than in leaves.

Conclusions

These results agree with the theoretical prediction that adaptation of photosynthesis to elevated CO₂ requires increased RuBP regeneration. Cotton bracts are readily available material for studying adaption to elevated CO₂.     

A rapid purification procedure for tetrodotoxin derivatives by high-performance liquid chromatography

A simple procedure for purification of tetrodotoxin (TTX) derivatives by high-performance liquid chromatography is described. Chemically oxidized TTX, C₁₁-nortetrodotoxin (nor-TTX), was purified and collected by reverse-phase chromatography. The separation of nor-TTX from unreacted TTX was excellent and recovery of nor-TTX was more than 90%. The isolated nor-TTX was further coupled with lysine, and the coupled product was purified again by high-performance liquid chromatography on a cation-exchange column. The separation of all compounds required less than 15 min. The uv monitoring at 230 nm allowed the detection of TTX derivatives at the 2- to 3-ng level.     

Anaerobic bacterial reductions of aldehydes and ketones: a rapid screening procedure

A semi-quantitative screening procedure is described whereby the abilities of cell-free extracts of bacteria to oxidize a selection of alcohols are rapidly disclosed. The activities displayed in the preliminary screen generally correlate well with the relative abilities of washed suspensions of these bacteria to reduce the corresponding aldehydes and ketones. The method therefore promises to be helpful in informing the choice of the most appropriate bacterium wherewith to effect the bioreduction of any particular ketone.     

Simple and rapid procedure for the purification of lipoprotein(a)

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein-like particle displaying strong athero-thrombotic properties. Highly purified Lp(a) is increasingly requested for standardization of Lp(a) measurements and for biological studies. Several procedures have been described for Lp(a) separation and purification but none of them appear completely suitable. We present here a procedure for Lp(a) purification based on sequential elutions after lysine-Sepharose affinity chromatography. We were able to identify four distinct subspecies of Lp(a) showing different affinity to ε-amino groups of lysine-Sepharose, simply by modifying molarity and pH of the eluents; the fraction obtained in highly purified state represented the major form and could be eluted with 0.5 M sodium phosphate buffer (pH 4.4). Advantages of this procedure are represented by simplicity, rapidity and final yield.     

A rapid and efficient procedure for transformation of intact Saccharomyces cerevisiae by electroporation

A rapid and efficient procedure is described for transforming Saccharomyces cerevisiae using electroporation to render intact cells permeable to DNA. The technique uses relatively low voltages and is particularly sensitive to low concentrations of plasmid DNA. At the highest voltage used (400 volts), the frequency of transformation increased with the amount of plasmid DNA between 25 ng and 100 ng. At higher concentrations of DNA (1-1.5 micrograms) electroporation yielded one-third to one-half the number of transformants obtained with a standard lithium acetate pretreatment. Because this method requires neither pretreatment of cells nor addition of polyethylene glycol (PEG), it has several advantages over currently used transformation procedures.     

The radioreceptor assay: a simple, sensitive and rapid analytical procedure

Radioreceptor assays (RRAs) are analogous in concept to radioimmuno assays. Characteristic to both methods is the saturable, specific, competitive and reversible ligand/receptor interaction. The RRA is simple, sensitive and reproducible and provides a degree of precision comparable to more sophisticated analytical techniques. Since RRAs require little specialized equipment, they can be used routinely by any laboratory engaged in biochemical research or, as an inexpensive exercise to teach the fundamentals of ligand binding and analytical pharmacology.