A novel,Golgi-Cox-based fluorescent staining method for visualizing full-length processes in primary rat neurons

The Golgi method, a well-known method used for staining whole dendrites and axonal trees of neurons, has been used widely for studying dendritic growth in vivo. Although detailed structural examination of neurons and their processes stained by the Golgi method has elucidated the complicated neuronal circuit, application of the method in cultured neurons has been unsuccessful to date.     

一种简单快速高分辨率的PAGE胶显带方法

尽管使用常规方法对聚丙烯酰胺胶(PAGE胶)进行DNA显带, 能获得高分辨率图片, 但常规方法操作步骤繁琐, 耗时较长。文章报道了一种PAGE胶DNA显带的改进方法, 分别使用改进的显带方法和常规的显带方法进行了PAGE胶的显影和比较。结果表明, 使用改进的显带方法得到的PAGE胶图片, DNA带型和背景具有更高的对比度, 因此具有更高的分辨率; 同时操作步骤少, 耗时短, 使用试剂少。这种方法在本实验室中已经完全代替了常规PAGE胶显带方法。     

Temperature gradient DNA-probe column chromatography: as tools for detection and purification of particular DNAs and RNAs

We have developed the temperature-gradient DNA-probe column chromatography as the new method for detecting and purifying particular DNAs or RNAs accurately. The method has high discrimination resolution, and can detect a single-base change in sample base sequences. The method also has high performances in purification of particular DNAs or RNAs, which has been demonstrated by purification of bovine mitochondria serine t-RNA. None of other method can succeed in complete purification of this t-RNA molecule. The present method can be applied not only to molecular biology as basic tools for purifying and picking-up particular sequences but also to the medical field as diagnostic tools.     

Method for rapid screening of pesticide mineralization in soil

A method has been developed for the analysis of (14)CO(2) evolution from the mineralization of (14)C-labelled organic compounds in soil samples. The new method is less space demanding and substantially cuts down laborious manual work compared to the traditional incubation bottle method used. Furthermore, the use of scintillation cocktail is largely reduced with the new method. In the new method, (14)CO(2) is trapped in filter paper held in the lid of a 20 ml glass vial by surface tension. The trapping solution used is Ca(OH)(2), which fixates CO(2) in the filter paper and the analysis of trapped (14)CO(2) is done using the Cyclone trade mark Storage Phosphor system. The lids are placed in a 32 well holder and exposed to a phosphor screen prior to scanning in a Cyclone trade mark scanner. The new filter method has been tested and compared to results obtained using the traditional method. The results show good agreement but due to a smaller capacity for CO(2) with the filter method compared to the traditional method, the interval between sampling has to be shorter using the filter method when the CO(2) development is high. The detection limits for the filter method is higher compared to the traditional method. With the filter method, the level of radioactivity has to exceed 300 dpm before detection is possible, while the same limit for the traditional method is around 30 dpm. On the other hand, the gas trapping faster and the efficiency is higher with the filter method.     

Method of radiocapacity factor assessment in study of cross adaptations of plants

Adaptation of plants to the action of gamma-irradiation and cadmium chloride has been studied in the experiments on water culture of maize plants. A new method of registration of the biological object state at the conditions of the stress-factor action - "the method of radiocapacity factor" based on the measurement of the culture medium radio-activity during the incubation process of vegetative objects has been developed. The method offered has allowed us to estimate in dynamics an integrated condition of the root system exposed to stimulating and inhibiting doses of acute gamma-irradiation. Application of the pattern of radio-adaptive response has allowed us to reveal it using both a conventional technique - by registering the values of growing parameters, and the radiocapacity factor method.     

The biolistic process

The biolistic process is a new process which employs high velocity microprojectiles to deliver substances into cells and tissues. It has been described in different ways and has been referred to as the particle gun method, the microprojectile method, the gene gun method, the particle acceleration method, the bio-blaster method. The inventors of the process have coined the term ‘biolistic’ (biological ballistics) to describe both the process and any associated apparatus used to shoot biological materials into living targets.     

The Use of RP-HPLC for measuring activation and cleavage of rFVIIa during purification

A reverse phase HPLC (RP-HPLC) method for analysis of recombinant factor VIIa (rFVIIa) has been developed. The method discriminates between different forms of recombinant FVII (rFVII). To obtain separation of these closely related molecules the method has been optimized with respect to gradient profile and temperature. The method has been used for optimization of purification processes and for kinetic studies. EVidence for autolytic cleavage was obtained. (c) 1995 John Wiley & Sons, Inc.     

利用FPLC系统结合自装层析柱纯化兔血清IgG

为了建立一种简便、成本低的纯化兔血清IgG的实用方法,采用FPLC系统结合实验室常规手段填充的Sephacryl-S200凝胶柱和DEAE-Sephadex A-50离子交换柱进行兔血清IgG的纯化,结果表明,在流速为0．5ml／min时,此方法可以纯化得到高纯度的兔IgG;脱盐柱Sephadex G-25重复实验证明此实验方法具有很好的稳定性;并且与商品化的层析柱相比,成本大为下降;此方法主要的不足之处是层析柱的流速较低,为0．5ml／min左右。该方法可以推广到其他生物大分子的纯化中去。     

Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E

A spectrophotometric method has been developed for the quantitative determination of antioxidant capacity. The assay is based on the reduction of Mo(VI) to Mo(V) by the sample analyte and the subsequent formation of a green phosphate/Mo(V) complex at acidic pH. The method has been optimized and characterized with respect to linearity interval, repetitivity and reproducibility, and molar absorption coefficients for the quantitation of several antioxidants, including vitamin E. The phosphomolybdenum method, in combination with hexane monophasic extraction, has also been adapted for the specific determination of vitamin E in seeds. The results obtained with the proposed method were validated by comparison with a standard HPLC method. The phosphomolybdenum method is routinely applied in our laboratory to evaluate the total antioxidant capacity of plant extracts and to determine vitamin E in a variety of grains and seeds, including corn and soybean.     

Comparison of gas chromatography mass spectrometry methods for the determination of delta9-tetrahydrocannabinol in plasma

A method for the identification of delta9-tetrahydrocannabinol by gas chromatography mass spectrometry has been developed, and this method has been compared with other techniques, such as detection via thin-layer chromatography using tritium labeled delta9-tetrahydrocannibinol and a dual gas chromatographic method. The gas chromatographic mass spectrometric method was found to be equal or superior to other techniques and has the added advantage of being highly specific for the compound analyzed. An alternate approach using chemical ionization is also described; however, this procedure does not show significant advantages over the electron impact method. These methods show a practical lower detection limit of 500 pg ml-1 of plasma in clinical practice.     

An easy-to-handle semi-automated method for media development using a colorimetric viability assay and fractional factorial designs

A rapid and effective semi-automated screening method has been developed for the development of growth media for mammalian cell culture. The method has proven to be a powerful tool for preliminary evaluation and comparison of new media formulations, but has some inherent disadvantages, which are important to recognise in order to interpret the results.     

Estimation of Incidence from Age Wise Prevalence

A method used by various researchers for estimation of incidence from prevalence data of a stable population for irreversible stable diseases has been validated. For this purpose a followup data was generated by life table distribution techniques with known mortality and incidence rates. The results show that the method as such cannot be used to estimate the true incidence from prevalence data of a crossectional study from stable population. The remote situation where the method will work has been discussed. A method has also been presented for situations where the known method fails i.e. duration of irreversible disease is less than the age of effected persons in a stable population.     

Diagnostic possibilities of magnetic resonance cholangiopancreatography

Studies for 39 patients have been done with the use of a new non-invasive method of observation--MR cholangiopancreatography,--that enables to get a high contrast-enhanced image of the intrahepatic, extrahepatic and pancreatic ducts without administration of the contrast agents and invasive interventions. High diagnostic efficiency of the method has been proved in patients with diseases of bile ducts and pancreas. The observation technique has been optimized and semiotics has been clarified in cases of various diseases that cause on obstruction of the biliferous tracts. The advantages of this method has been showed as compared to invasive roentgenologic methods of examinations.     

A new assay method for hydrogenase based on an enzymic electrode reaction. The enzymic electric cell method.

A new assay method for hydrogenase [EC 1.12.2.1] based on the enzymic electrode reaction of H2-H+ equilibrium has been established. The method is based on the experimental fact that the short-circuit current of the electric cell composed of an electrode with hydrogenase and methylviologen as the mediator of H2-H+ equilibrium and a saturated calomel electrode as the counter electrode, is practically proportional to the amount of hydrogenase in the cell. The new method is referred to as the "enzymic electric cell method." This technique has applications not only to routine activity assay but also to the direct determination of the time course of enzyme denaturation, which has not previously been possible.     

Analysis of ligand-receptor binding by the difference method

A new method has been proposed for analysis of experimental data on ligand-receptor binding at equilibrium. This method makes it possible to detect heterogeneity of a receptor system in cases where the contribution of the high-affinity site to total binding is rather small and the problem of graphic discrimination of a model cannot be solved unambiguously by other methods. The difference method permits us to exclude experiments on measuring nonspecific binding. A computer program for analysis of ligand-receptor binding has been worked out in which the difference method and traditional methods of binding isotherm analysis are realized. Numerical modeling has shown that the best strategy in experimental data processing is the treatment of total binding isotherms by both the difference method and regression analysis, including the nonspecific binding constant as one of the regression parameters.     

A method for the indirect determination of trace bound selenomethionine in plants and some biological materials

An indirect method for the determination of trace bound selenomethionine (SeMet) has been developed. SeMet reacts with cyanogen bromide (CNBr) quantitatively in the presence of SnCl2 to form CH3SeCN, and after extraction with CHCl3 is acid-digested to form Se(IV). Selenium(IV) reacts with 4-nitro-o-phenylenediamine reagent to form 5-NO2-piazselenol which is then determined by gas chromatography equipped with electron capture detector. The sensitivity of this method (CNBr-piazselenol-GC method) is 6 ng SeMet/g of sample. Trace-bound SeMet in plants and some biological materials has been successfully determined by this method and its content has been compared with the total selenium in the sample.     

Determination of aminoethylcysteine ketimine decarboxylated dimer in human plasma and cultured cells by high-performance liquid chromatography with electrochemical detection

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) is a natural compound with antioxidant properties of a new family of sulfur-containing amino acids. It has been detected in human urine and plasma, in mammalian cerebellum and, more recently, in dietary vegetables. In the present study, a simple, highly sensitive method using a high-performance liquid chromatography system with electrochemical detection (ECD) has been developed. The method showed excellent precision and accuracy. It has been found to be about 100-fold more sensitive than gas chromatographic method and 2000-fold more sensitive in respect to the liquid chromatography method with UV detection. The method showed the required features of specificity and sensitivity to detect aminoethylcysteine ketimine decarboxylated dimer in human plasma and in cultured cells after in vitro supplementation.     

Random forest for gene selection and microarray data classification

A random forest method has been selected to perform both gene selection and classification of the microarray data. In this embedded method, the selection of smallest possible sets of genes with lowest error rates is the key factor in achieving highest classification accuracy. Hence, improved gene selection method using random forest has been proposed to obtain the smallest subset of genes as well as biggest subset of genes prior to classification. The option for biggest subset selection is done to assist researchers who intend to use the informative genes for further research. Enhanced random forest gene selection has performed better in terms of selecting the smallest subset as well as biggest subset of informative genes with lowest out of bag error rates through gene selection. Furthermore, the classification performed on the selected subset of genes using random forest has lead to lower prediction error rates compared to existing method and other similar available methods.     

Multimode Optical Fiber Surface Plasmon Resonance Signal Processing Based on the Fourier Series Fitting

Multimode optical fiber is widely used because of its large core size, strong light-gathering ability, and remote on-line sensing ability in various environments. Due to the mode coupling and polarization loss, the signal curve obtained has wide full width at half-maximum (FWHM) and small peak value. Therefore, efficient methods to calculate the resonant wavelength are required. This paper presents a method to process the surface plasmon resonance curve (SPR) based on the Fourier series fitting. The calculated resonant wavelength is obtained with one-dimensional extremum-search method. Experiment results show the proposed method can accurately calculate the resonant wavelength of high concentration solution. Additionally, the method has higher sensitivity and is unaffected by the light source fluctuation. This method has potential value in processing the SPR signal of the multimode optical fiber.     

Development of a carbon dioxide-capture assay in microtiter plate for aspartyl-beta-hydroxylase.

CO2-capture methods have been used for assaying many decarboxylating enzymes including hydroxylation-coupled decarboxylation reactions. The traditional CO2-capture method involves performing the reaction in capped tubes and radiometric measurement of trapped 14CO2 by scintillation counting. In this report, a 14CO2-capture method in a 96-well microtiter plate format has been developed and a phosphor imaging system has been employed for sample measurement. The new assay method has been used successfully to assay aspartyl-beta-hydroxylase activity in microtiter plate format. The results obtained here compare favorably with those obtained from the traditional tube method. The method is sensitive, suitable for high throughput, and generally applicable to many CO2-releasing enzyme assays.