A new assay for endocytosis in erythrocyte ghosts based on loss of acetylcholinesterase activity

A new method for assaying endocytosis in erythrocyte ghosts is presented. The method involves measuring the percentage loss of acetylcholinesterase activity which occurs when vacuoles form, making the acetylcholinesterase on the vacuole surface inaccessible. This method is compared to other methods of measuring endocytosis in this system, including phase contrast microscope estimation of vesiculation, stereological analysis of electron micrographs to determine vesiculation and loss of sialic acid accessible to neuraminidase due to endocytosis. Comparison of the percentage loss of acetylcholinesterase activity with the electron micrographic and sialic acid methods showed that all three methods gave a quantitative measure of the percentage of total membrane area taken in as vesicles. Since the acetylcholinesterase method was fast, easy, inexpensive, and quantitative, it was the preferred method for assay of endocytosis. The inhibition of endocytosis by Ca2+ was observed with this method; the success of this experiment demonstrated the applicability of the method to the study of inhibitors of endocytosis.     

A new oral contraceptive based on the normophasic method

62 women were treated for 554 cycles with normophasic oral contraceptive Fisioquens. A treatment cycle consists of 7 tablets of 0.05 mg ethinyloestradiol, followed by 15 tablets with a combination of 0.05 mg ethinyl-oestradiol and 1 mg lynestrenol. No pregnancies occurred. Both tolerance of the preparation and cycle control were good. Irregular bleeding occurred sporadically. Various side-effects diminished during treatment and even disappeared completely. Fisioquens appears to be a reliable contraceptive with a minimum of side-effects.     

A new method for diffusion measurement in polymeric films based on a stacked sheet concept

PURPOSE: To develop and evaluate a simple, yet well defined, method to measure diffusion in semi-solids, i.e. polymeric materials. METHODS: The method was based on a concept where equivalent discs of polymeric films were cut and stacked on top of each other. The diffusion process was allowed to proceed unidimensionally through the stack of films perpendicular to the film surface. After an appropriate time, the stack was analysed disc by disc with respect to solute content and from the concentration profile so obtained the diffusion coefficient was calculated. RESULTS: An all-in-one device was developed, manufactured in stainless-steel, that cuts circular discs and stores each one successively in a "stack" in the cell compartment. CONCLUSIONS: Data from a silicone based system shows that the method, although simple, is accurate and reproducible.     

Evaluation of a new method for identification of bacteria based on sequence homology of 16S rRNA gene

A new identification method for bacteria based on partial sequences of divergent regions of the 16S rRNA gene was evaluated. The method involves PCR-based amplification of 16S rRNA gene fragments, followed by sequencing and comparison of sequences of about 300 nucleotides with those in the database of NCBI (National Center for Biotechnology Information) via the Internet. Most of the bacteria tested could be identified at the species level even if some unread nucleotides were present in the sequence. Although this method still requires improvement, it has the potential to be a highly reliable and practical identification method for bacteria.     

基于固有振动频率的滑坡安全评价新方法

运用模型实验,在保持下滑力不变的情况下,通过固有振动频率对滑坡内部的黏结力、摩擦力等抗滑力指标进行分析.通过在弱稳定阶段中实际静摩擦力是否达到最大静摩擦力的方法,科学地判识滑体的稳定情况.结果表明:计算的摩擦力可以有效分析滑坡在弱稳定阶段期间的安全性,并证明固有振动频率监测比位移监测更加敏感.同时,固有振动频率的监测可对滑坡损伤做出定量判断,并可以评估滑坡静摩擦力指标,从而实现扰动后滑坡的安全评价.     

A colorimetric assay method for the evaluation of neurotrophic activity in vitro

A colorimetric assay was established to detect neurotrophic activity by measuring the lysosomal enzyme, acid phosphatase (AP) activity of cultured neuronal cells. Neurons from the cerebral cortex of 14- or 15-day mouse embryo were cultured in serum-free medium for 3 days in 96-well culture plates. A linear relationship was obtained between the AP activity and the number of viable neurons counted under a microscope. The AP assay was used to evaluate the neurotrophic activity of basic fibroblast growth factor. This assay is shown to be simple, sensitive and convenient to detect neurotrophic activity.     

基于辐射图像处理技术的保护渣熔化速度测量新方法

利用保护渣熔化过程中颗粒数量不断减少,完全熔化后颗粒完全消失的特征,提出了一种基于辐射图像处理技术的保护渣熔化速度测量方法。该方法采用机器视觉和图像处理技术,实现了保护渣熔化速度在线测量,并利用机器视觉库OpenCV实现了保护渣辐射图像中的中值滤波、图像分割、边缘检测和颗粒统计等图像处理功能并开发了测试软件。现场应用表明,以保护渣颗粒为检测信息的熔化速度测试理论及技术是可行的,与传统检测方法相比,新方法能定量检测保护渣的熔化速度并能实现熔化过程可视化监控。     

Measurement of antibody/antigen association rate constants in solution by a method based on the enzyme-linked immunosorbent assay

A reliable ELISA based method has been developed for measuring in solution antigen/antibody association rate constants. Its rationale is as follows: antigen and antibody are mixed in solution to initiate the association. At different time intervals aliquots are withdrawn to determine by an indirect ELISA the amount of free antibody that remains in solution. The disappearance of the free mAb reflects the time course of the association reaction. To test the validity of this method, the association rate constant of a monoclonal antibody for its antigen was measured and compared with that obtained previously by a method using fluorescence. The good agreement between the results obtained with the ELISA-based method and those obtained previously by fluorescence measurement indicates that the method described permits determination of true association rate constants in solution. The present method offers several advantages. It uses only minute amounts of sample which need not be purified; it requires no radioactive or fluorescent labelling of the antibody or the antigen, and it can be applied to any type of complex between macromolecules if an ELISA test can be set up to detect quantitatively one of the macromolecules.     

利用碳钢铸坯组织灰度图获取C含量分布的方法

摘要：基于热酸洗腐蚀原理与图像光学理论,提出运用金属低倍组织灰度分析技术（灰度分析法）获取中高碳钢铸坯的低倍组织中不同位置不同区域不同尺度的一维、二维C元素含量分布。偏析严重的位置得到的C元素含量高,初始凝固的晶粒中心位置得到的C元素含量低,说明通过灰度分析法计算的C元素含量分布能与低倍组织形貌很好地对应。同时,通过电子探针与灰度分析法的测量结果对比,发现二者C元素含量的变化趋势具有很好的一致性,这验证了灰度分析法的有效性。另外,低倍组织图像整体亮度的改变不会造成由灰度分析法所测量的C元素含量发生明显改变,则表明灰度分析法对测量环境的变化具有一定的抗干扰能力,也间接说明了其可靠性。     

A strain of the yeast Saccharomyces cerevisiae for testing environmental mutagens based on interaction of the mutations rad2 and him1

The interaction between mutations at the RAD2 and HIM1 genes was studied. The RAD2 gene encodes endonuclease involved in nucleotide excision repair. Mutants at this gene are highly sensitive to the lethal effect of a variety of mutagens. The product of the HIM1 gene is needed for correction of mismatched bases and repair of premutational DNA damage. Mutations in this gene lead to the formation of the mutator phenotype and high sensitivity to induced mutagenesis. The double rad2 him1 mutant manifested the synergic type of interaction. The level of UV-induced mutagenesis in the double mutant was five times higher than in single mutants, and the absolute yield of forward mutations in five genes controlling adenine biosynthesis was 1 to 2%. UV-induced mutagenesis was increased, at low doses, by several orders of magnitude in the double mutant, compared to the wild-type strain. The high level of mutagenesis in this mutant was caused by ethyl and methyl methanesulfonate. These properties of the stock with the double rad2 him1 mutation makes it promising as a tester in analysis of the gene toxicity of different substances.     

A simple method for the assay of eight steroids in small volumes of plasma

A simple method is described for the simultaneous radioligand assay of four delta5-3beta-hydroxysteroids adjacent to one another on the biosynthetic pathway (pregnenolone [1], 17alpha-hydroxypregnenolone, dehydroepiandrosterone and 5-androsterone-3beta, 17beta-diol), and their four delta4-3keto products (progesterone, 17alpha-hydroxyprogesterone, 4-androstene-3, 17-dione and testosterone). Two plasma aliquots are extracted and fractionated each for four steroids and individual corrections are made for losses. For fractionation, maximum use is made of the high resolution and reproducibility of celite minicolumns, using propylene glycol as stationary phase, and a discontinuous gradient of ethyl acetate in iso-octane as mobile phase. The fractions are then assayed in the appropriate radioligand end-assay system. Each assay was finally validated by demonstrating coincidence of peaks of immuno- and radioactive steroid in extracts of female plasma. Results in pre-pubertal girls and women in the follicular phase of the menstrual cycle suggest that the major change in adrenal steroid production at puberty may be an increase in 17, 20-desmolase activity. There appears to be little reversal of this change in adrenal function after ovariectomy.     

A comprehensive evolutionary analysis based on nucleotide and amino acid sequences of the alpha- and beta-subunits of glycoprotein hormone gene family

On the basis of nucleotide sequences of the coding region and their predicted amino acid sequences, 58 glycoprotein hormone subunit genes were compared, aligned and used to construct phylogenetic trees for this family. The analysis included 17 alpha-subunits, eight TSH beta-, six FSH beta-, 17 LH beta/CG beta-, four fish gonadotropin (GTH)-I beta-, five fish GTH-II beta- and one additional fish GTH beta-subunit. The reliability of the phylogenetic trees was probed with the bootstrapping test. Our results indicated that: both the alpha- and beta-subunits of the family diverged from a common ancestral gene about 927 million years ago, the initial precursor of the beta-subunit duplicated to give rise to the LH beta and a second hormone, the latter then duplicating to FSH beta and TSH beta, so that FSH beta is related more to TSH beta than to LH beta; and bony fish GTH-I beta is highly related to mammalian FSH beta, whereas the bony fish GTH-II beta is more related to mammalian LH beta. For scientific consistency and convenience, we propose that the following nomenclature be adopted, all fish gonadotropins of type I be classified as FSH and all type II be classified as LH hormones. In addition, on the basis of results from this and other studies, we propose an evolutionary history for this glycoprotein hormone family. Reconstruction of the evolutionary history of this family would not only provide clues to understanding thyrotropin and gonadotropin functions, but would also allow further revision of the present nomenclature of the gonadotropins in fish.     

A new mumps virus lineage found in the 1995 mumps outbreak in western Switzerland identified by nucleotide sequence analysis of the SH gene

We determined the nucleotide sequence of the SH gene its flanking regions over a range of 380 nucleotides for three distinct mumps virus (MUV) isolates. Two isolates from the 1992 mumps epidemic in Western Switzerland and one MUV isolated in 1995 in the same geographic area have been analyzed and compared to 16 recently published SH nucleotide sequences and their presumed amino acid sequences. The nucleotide sequences from the 1992 MUV isolates were identical and closely related to two MUV strains from Eastern Switzerland and strains from the U.K. The MUV isolated in 1995 is clearly different from all other strains.     

A combination of a common splice site mutation and a frameshift mutation in the COL7A1 gene: absence of functional collagen VII in keratinocytes and skin

Musk xylene (2,4,6-trinitro-1-t-butylxylene; MX) is a synthetic nitromusk perfume ingredient that induces and inhibits mouse cytochrome P4502B (CYP2B) enzymes in vivo. The purpose of the present work was to determine whether amine metabolites of MX contributed to the enzyme inhibition and, if so, to define the nature and kinetics of this inhibition. When dosed orally to phenobarbital (PB)-treated mice, MX (200 mg/kg) inhibited > 90% of the PB-induced O-dealkylation of 7-pentoxyresorufin (PROD), and [14C]MX equivalents bound covalently to microsomal proteins. However, when this experiment was repeated in mice pretreated with antibiotics to eliminate the gastrointestinal flora, no decrease in PB-induced PROD activity and no covalent binding to microsomal proteins were observed. Thus, the ability of antibiotic treatment to eliminate the enzyme inhibition and covalent binding implicated amine metabolites of MX formed by nitroreduction in anaerobic intestinal flora as obligatory for these effects. Two monoamine metabolites of MX were synthesized to study enzyme inhibition directly. These metabolites were 2-amino-4,6-dinitro-1-t-butyl-xylene and 4-amino-2,6-dinitro-1-t-butylxylene, referred to as o-NH2-MX and p-NH2-MX, respectively, reflecting the position of the amine substitution relative to the t-butyl function. In the in vitro studies with PB-induced mouse liver microsomes, both amines inhibited PROD activity when preincubated in the absence of NADPH. However, only p-NH2-MX caused a time- and NADPH-dependent loss of PROD activity, and the inactivation rate was a pseudo-first-order process that displayed saturation kinetics. These results indicate that p-NH2-MX is a mechanism-based inactivator of mouse CYP2B enzymes. From kinetic analyses, the Ki was calculated to be 10.5 microM and the Kinact was 1.2 min-1. As final confirmation of the inhibitory effects of p-NH2-MX on mouse CYP2B enzymes, the amine (0.67 mmol/kg) was dosed orally to PB-induced mice. At 2 hr after dosing, p-NH2-MX inhibited essentially all of the PB-induced PROD activity, whereas an equimolar dosage of parent MX had no effect at this early time. Thus, although MX is an inducer of mouse CYP2B enzymes, an amine metabolite of MX is a mechanism-based inactivator of mouse CYP2B10. Furthermore, it is likely that the amine is responsible for the lack of functional CYP2B enzyme activity associated with induction of this enzyme by MX.     

Glycated haemoglobin in various pathological conditions: investigations based on a new, fully automated method

A new automated affinity chromatographic method for determining glycated haemoglobin (GHb) in dogs and cats was tested. The method appeared to be practical, quick and accurate. The reference range, calculated on the basis of 50 healthy dogs and 43 healthy cats, lay between 2.4 and 3.4 per cent in dogs and 2.0 and 2.9 per cent in cats. Concentrations were not influenced by age or gender. GHb levels obtained for 21 dogs and 18 cats with newly diagnosed diabetes mellitus were significantly higher than those of the control animals, ranging from 4.5 to 8.6 per cent (median 6.1) in dogs and from 2.7 to 6.0 per cent (median 3.8 per cent) in cats. The GHb levels in 31 normoglycaemic dogs with anaemia ranged from 2.3 to 4.3 per cent (median 3.3 per cent), and those of 22 normoglycaemic cats with anaemia from 2.6 to 3.9 per cent (median 3.2 per cent); both sets of levels were significantly elevated compared to control group values. GHb concentrations in animals with polycythaemia, azotaemia or liver disease showed no significant deviations from the control group; in individual cases they were slightly elevated compared to the reference range. The automated measuring method employed can be used to determine GHb in dogs and cats. Anaemic animals should generally be excluded from the GHb determination.