Directed mutagenesis of YAC-cloned DNA using a rapid, PCR-based screening protocol

We have developed a system which facilitates the rapid modification of yeast artificial chromosome (YAC) insert DNA. Specific modifications, such as deletions, insertions and point mutations, can be generated by a two-step allele replacement method using the yeast translational suppressor, SUP4-o, as both a positive and negative selection. The introduction of the SUP4-o gene was successful in 4 out of 24 selected transformant colonies, while the subsequent homologous elimination occurred in 2 out of 30 colonies. The use of a simple, short-range PCR assay rapidly identified the correct events among the genetically selected isolates and should be generally applicable to YAC modifications.     

A PCR-based strategy for extensive mutagenesis of a target DNA sequence

A mixed population of mutagenic oligonucleotide primers was used to generate a set of point mutations in a short region of a retroviral gene by PCR amplification. The mixed population of mutagenic primers was generated by incorporating a mixture of A, G, C and T at specific sites during oligonucleotide synthesis. With the proportions of mutagenic nucleotides used for our experiments, 47 percent of the 213 clones analyzed had one or more point mutation in the target DNA sequence. In addition, unpredicted mutations were observed that contributed to the mutagenic complexity of the population. We have found this approach to be an efficient means for extensive mutagenesis of a defined target DNA sequence.     

An improved method for detecting passive pills

One of the principal disadvantages of the passive pill as a telemetric method for measuring various physiological parameters has been its resticted range. The reasons for the restricted range with existing detection methods are discussed. An improved method using a locking spectrometer based on third-order phase-sensitive detection is described and its performance is assessed. A significant increase in the usable range of a high sensitivity passive pill is obtained.     

An improved assay method for fibroblast gelatinolytic enzyme

A useful gelatinolytic enzyme assay for fibroblasts, utilizing a novel sample preparation method for collagenase with dithiothreitol (DTT) treatment to inactivate endogenous collagenase inhibitors, was developed using soluble fluorescein isothiocyanate (FITC)-labeled gelatin. The substrate, gelatin was prepared by heating commercially available FITC-labeled type I collagen. The denatured collagen was cleaved with purified trypsin and partially purified fibroblast gelatinase, and the digested FITC-fragments were measured fluorometrically. The intensity of the fluorescence was in proportion to the reaction time and enzyme concentration. Both enzyme activities were measurable within the nanogram range of enzyme preparations. The enzyme activity was detected after 4-aminophenylmercuric acetate (APMA) treatment which was completely inhibited by metalloproteinase inhibitors, but not by serine- and cysteine-proteinases' inhibitors. Conditioned media of human periodontal ligament fibroblasts (PLF) and gingival fibroblasts (GF) were separately treated with DTT prior to the enzyme assay, and then the assay was performed in the presence of APMA. The enzyme activities of PLF and GF were 106- and 55-fold higher than those of the conventional gelatinase assay which was carried out without DTT treatment. This assay method allowed the measurement of gelatinolytic enzyme activity when tissue inhibitors of metalloproteinases were present in the fibroblast culture medium.     

An improved UV-spectrophotometric method for routine barbiturate monitoring

Clinical tests, for instance pupillometry, Valsalvatest, and orthostatic tests show evidence of sympathicopathy in chronic renal failure (GFR: less than 30 ml/min). Hemodialysis does not improve the intensity or frequency of the alterations. Toxic effects of uremic toxins on the autonomic nervous system can be supposed.     

An improved method for chromosome counting in maize

An improved method for counting chromosomes in maize (Zea mays L.) is presented. Application of cold treatment (5C, 24 hr), heat treatment (42 C, 5 min) and a second cold treatment (5C, 24 hr) to root tips before fixation increased the number of condensed and dispersed countable metaphase chromosome figures. Fixed root tips were prepared by the enzymatic maceration-air drying method and preparations were stained with acetic orcein. Under favorable conditions, one preparation with 50-100 countable chromosome figures could be obtained in diploid maize using this method. Conditions affecting the dispersion of the chromosomes are described. This technique is especially useful for determining the somatic chromosome number in triploid and tetraploid maize lines.     

混匀矿粉采样技术的改进与应用

通过对混匀矿粉采样技术的改进，解决了以往混匀矿粉采样缺乏代表性的难题，实现了混匀矿粉按预算成份组织生产的目的，为其他散状物料的采样提供了一定的经验．     

An improved automated fluorimetric method for determination of histamine

The automated continuous flow system for the extraction and fluorimetric analysis of histamine based on the principle of Shore et al. (1959) has been improved. With lower consumption of reagents and further simplification of the working conditions, histamine can be determined quantitatively in a routine fashion in aqueous samples, with or without protein content, up to a concentration of approximately 0.1 ng/ml. The rate of analysis is 30 samples/h.     

黑芥子酶提取及活性测定方法的改进

以拟南芥为材料,对以往黑芥子酶的提取方法和活性测定方法进行了改进,通过实验优选出最佳的提取和活性测定条件.改进后的方法提取样品量只有150 mg,提取缓冲溶液1 mL,离心回收上清作为酶液;3 mL反应体系溶液(含0.2 mmol/L黑芥子硫苷酸钾)置于1 cm光路石英比色杯中,37℃条件下平衡后,向反应体系溶液中添加150μL酶液,预反应3min,而后在227 nm波长下测量吸光度5 min(反应时间),计算酶活.改进后的方法取样量小,操作较为简便,适于较多样品的平行测定.     

An improved method for deriving equal-discriminability scales from ratings.

Rating scales derived with variability of judgment as the unit of measurement contain a spurious factor arising from differences in the constant rating tendencies of individuals. A technique for constructing scales which yield distributions as nearly as possible equal in dispersion is illustrated. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by using site-directed mutagenesis

The actin-activated ATPase activity of Acanthamoeba myosin IC is stimulated 15- to 20-fold by phosphorylation of Ser-329 in the heavy chain. In most myosins, either glutamate or aspartate occupies this position, which lies within a surface loop that forms part of the actomyosin interface. To investigate the apparent need for a negative charge at this site, we mutated Ser-329 to alanine, asparagine, aspartate, or glutamate and coexpressed the Flag-tagged wild-type or mutant heavy chain and light chain in baculovirus-infected insect cells. Recombinant wild-type myosin IC was indistinguishable from myosin IC purified from Acanthamoeba as determined by (i) the dependence of its actin-activated ATPase activity on heavy-chain phosphorylation, (ii) the unusual triphasic dependence of its ATPase activity on the concentration of F-actin, (iii) its Km for ATP, and (iv) its ability to translocate actin filaments. The Ala and Asn mutants had the same low actin-activated ATPase activity as unphosphorylated wild-type myosin IC. The Glu mutant, like the phosphorylated wild-type protein, was 16-fold more active than unphosphorylated wild type, and the Asp mutant was 8-fold more active. The wild-type and mutant proteins had the same Km for ATP. Unphosphorylated wild-type protein and the Ala and Asn mutants were unable to translocate actin filaments, whereas the Glu mutant translocated filaments at the same velocity, and the Asp mutant at 50% the velocity, as phosphorylated wild-type proteins. These results demonstrate that an acidic amino acid can supply the negative charge in the surface loop required for the actin-dependent activities of Acanthamoeba myosin IC in vitro and indicate that the length of the side chain that delivers this charge is important.     

An improved method for the production of antibodies to lipophilic carboxylic hapten using small amount of hapten-carrier conjugate

Normal human diploid cells have a limited proliferative lifespan in in vitro cultures. Changes in gene expression have been examined for understanding control mechanisms of limited proliferative lifespan. and enhanced expression of growth suppressing genes such as p21 was reported in late-passaged cells. We screened genes which were expressed preferentially in mid-passaged cells by the differential plaque screening of the subtracted cDNA libraries prepared from young, life-extended, and immortalized SV40-transformed human fibroblasts. Among isolated clones, ASF/SF2, which was known to affect alternative splicing, was expressed in normal fibroblasts with a peak at mid-passage. Relative expression levels of SC35 and hnRNPA1, which are also known to affect alternative splicing, was also highest at mid-passage. Changes in alternative splicing at mid-passage, if it occurred, may play a crucial role in the process of cellular senescence.     

An improved method of intralamellar keratoplasty in rats

Intralamellar keratoplasty in inbred rats is a valuable method for the investigation of corneal allograft rejection. Present methods of intralamellar corneal grafting are tedious and time-consuming. In addition, induction of neovascularization, a prerequisite to inducing graft rejection, is variable. In an effort to overcome these problems, the authors have developed an improved method of intralamellar keratoplasty. Intralamellar pockets are formed by introducing a 30-g needle into the corneal stroma near the limbus. Approximately 10-25 microliters of lipopolysaccharide (LPS) (100 micrograms/ml) is injected into the stroma, forming a stromal bleb. This bleb is incised to form a pocket, resulting in the leakage of the injected liquid. The intralamellar pocket formation is completed by dissecting any remaining stromal fibers in the area of former bleb. Once the pocket has been formed, grafts of corneal tissue are inserted, and the incision is closed. This method of keratoplasty has the following advantages over previously reported methods: (1) It is more rapid and less tedious because the formation of the corneal bleb protects the anterior chamber from being invaded during the corneal incision; (2) It leads to a reproducible induction of neovascularization in every cornea so treated; (3) It results in a higher frequency of allograft rejection.     

An improved assessment method for the temperature dependence of yield strength values

In Europe work is in progress to establish new standards for materials and it is very important that accurate data are used as a basis for property values in these standards. In addition reliable evaluation methods must be employed when the values are derived. A systematic evaluation method for yield strengths at elevated temperatures has been developed which is a modification of the ISO 2605/111 (ENV22605-3) procedure. The method has been applied to a number of steel types and has proven to provide values in good accordance with experimental data. Comparisons with existing national and international standards showed the importance of using experimental values when establishing new standards. The method is intended for use in the standard developments by the European Committee for Iron and Steel Standardisation (ECISS).     

An improved method for rapid and efficient radioiodination of iodine-123-IQNB

The SPECT radioligand, 3-quinuclidinyl-4-[123I]iodobenzilate ([123I]IQNB), binds to muscarinic receptors and has generated interest as a potential agent for clinical SPECT. Unfortunately, cumbersome and inefficient radioiodination procedures have limited the practicality of [123I]IQNB SPECT imaging. METHODS: We report a rapid (5 min) and simple radioiodination procedure for preparing [123I]IQNB from a tri-n-butylstannyl precursor in a no-carrier-added reaction that yields high specific activity with radiochemical yield exceeding 60%. The radiochemical purity of the final product exceeds 95%. RESULTS: We have used this procedure to radioiodinate the four stereoisomers of [123I]IQNB. The procedure is highly reliable and reproducible. SPECT studies on a healthy human volunteer at 1, 2, 6 and 24 hr after injection of each of the four stereoisomers reveal expected differences in the kinetic and binding characteristics of the four stereoisomers. (R,S)-[123I]IQNB appears to be the SPECT agent of choice. CONCLUSION: Radioiodination of [123I]IQNB from our tri-n-butylstannyl precursor is simpler, more efficient and less expensive than previous techniques. The potential exists for a "kit" which would be practical in a typical clinical setting.