An enzymatic method for the determination of the initial rate of cholesterol esterification in human plasma.

A method is described for the determination of the initial rate of cholesterol esterification in human plasma, based on the enzymatic determination of free cholesterol in the plasma before and after incubation at 37 degrees C. The cholesterol esterification rate was linear up to 40 minutes. In 18 normal male and 10 normal female subjects the cholesterol esterification rate was 91 +/- 15 (mean +/- SD) and 62 +/- 12 nmoles/hr/ml of plasma, respectively.     

测定菌体浓度的简便方法

为了简便地测定测定JIS L 1902标准所规定的菌液浓度,以单位体积菌落数与吸光度(ABS值)的相关性,制成标准曲线,利用标准曲线快速准确地判断菌液浓度。     

An electrometric method for the determination of tyrosinase activity.

The pathway of dopachrome formation from L-dopa involves the net release of one proton for each molecule of dopachrome formed. The protons produced as a consequence of the enzymic step catalysed by tyrosinase can be measured by an electrometric device able to monitor changes in H+ concentration below 1 microM. This electrometric recording can be used as a simple, sensitive and continuous method for determining tyrosinase activity. The electrometric method can also be used in the presence of ascorbate by the spontaneous coupling of ascorbate oxidation to dopaquinone reduction, but measuring proton uptake instead of proton release.     

An easy cAMP extraction method facilitating adenylyl cyclase assays.

We present a facile procedure for measuring adenylyl cyclase activity which circumvents the two-step chromatographic purification of 32P-labeled cAMP. cAMP produced by stimulated cell membrane preparations is easily purified by organic extraction and thus available for quantification using tritium-labeled tracer cAMP and commercially available cAMP-binding protein. The quantification of cAMP is unaffected by the extraction procedure and sample handling. Data obtained by this method were identical to those obtained by the chromatographic method. This procedure could be shown to be suitable for measuring receptor-mediated adenylyl cyclase modulation.     

An easy, quantitative method for detection of endonuclease activity

An easy yet sensitive assay has been developed for the detection of endonuclease activities. The method involves the use of agarose gel electrophoresis to resolve intact homogeneous nucleic acid substrate from degradation products resulting from a small number of nucleolytic breaks. The assay is quantitative when a radioactively labeled nucleic acid is used as substrate, and it is as sensitive in the measurement of nuclease activity as is zone sedimentation in sucrose gradients. The assay can detect as few as 1.4 nicks, on the average, per substrate molecule. Its advantage over previous methods of analysis is the ease with which large numbers of samples can be handled while still retaining a high degree of sensitivity. The method is demonstrated with single-stranded DNA substrate, but it can be easily modified to detect endonuclease degradation of double-stranded DNA or degradation of RNA substrate.     

Two-dimensional gels: An easy method for large quantities of proteins

A new two-dimensional gel technique has been developed that makes it possible to load large amounts of protein and still retain good resolution. Protein samples are added to the isoelectric-focusing acrylamide solution prior to polymerization. Focusing times are short, making it possible to complete both dimensions of a two-dimensional gel in five to six hours. The technique yields larger amounts of separated protein for sequencing, and also greatly increases the quality of two-dimensional immunoblots.     

An easy method for cutting and fluorescent staining of thin roots

Background and Aims

Cutting plant material is essential for observing internal structures and may be difficult for various reasons. Most fixation agents such as aldehydes, as well as embedding resins, do not allow subsequent use of fluorescent staining and make material too soft to make good-quality hand-sections. Moreover, cutting thin roots can be very difficult and time consuming. A new, fast and effective method to provide good-quality sections and fluorescent staining of fresh or fixed root samples, including those of very thin roots (such as Arabidopsis or Noccaea), is described here.

Methods

To overcome the above-mentioned difficulties the following procedure is proposed: fixation in methanol (when fresh material cannot be used) followed by en bloc staining with toluidine blue, embedding in 6 % agarose, preparation of free-hand sections of embedded material, staining with fluorescent dye, and observation in a microscope under UV light.

Key Results

Despite eventual slight deformation of primary cell walls (depending on the species and root developmental stage), this method allows effective observation of different structures such as ontogenetic changes of cells along the root axis, e.g. development of xylem elements, deposition of Casparian bands and suberin lamellae in endodermis or exodermis or peri-endodermal thickenings in Noccaea roots.

Conclusions

This method provides good-quality sections and allows relatively rapid detection of cell-wall modifications. Also important is the possibility of using this method for free-hand cutting of extremely thin roots such as those of Arabidopsis.     

An easy method for the removal of Epon resin from semi-thin sections. Application of the avidin-biotin technique

Summary A simple procedure is described for removing Epon resin from semi-thin 1 m sections, which permits excellent postembedding immunohistochemical staining (avidin-biotin complex technique). The procedure was developed for the detection of growth hormone and prolactin in bovine adenohypophysis fixed with 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 m sodium cacodylate buffer pH 7.4–7.6. The results indicate that the removal of the epoxy embedding medium prior to the application of the immunohistochemical reagents was essential for the successful localization of the antigenic determinants of the two hormones. The immunocytochemical reactivity was obtained only after treating the sections with a solution of potassium hydroxide in a mixture of absolute methyl alcohol and propylene oxide (Maxwell's solution). An enhanced immunoreactivity was obtained when this treatment was followed by an additional treatment with either 4% hydrogen peroxide or a saturated aqueous solution of sodium metaperiodate. Because of the easy preparation of the Epon removal solution and the good structural preservation without damage to the antigenic determinants, Maxwell's solution is suggested as a good etching agent which can be used in immunohistochemical studies on semi-thin sections with excellent results.     

An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines.

The use of 2,4,6-trinitrobenzenesulfonic acid (TNBS) as a reagent for determining the concentrations of amines has been widely accepted (1–3) since its introduction in 1960 by Satakeet al. (4). The original procedure has since been modified by Mokrasch (5) to permit the determination of amines, amino acids, and proteins in mixtures. In both procedures the trinitrophenylation reaction is followed by a quenching step, after which the amino content is related to the increase in absorbance at 340 nm (4) or 420 nm (5). We have studied the trinitrophenylation reaction and have found that amino content can be related directly to the absorbance of the trinitrophenylation reaction mixture after a relatively short incubation period (15–30 min). Therefore, it is unnecessary to quench this reaction. We describe herein an extremely convenient procedure for the determination of amines, amino acids, and proteins where the quenching step employed by previous investigators has been eliminated. The proposed method has a greater sensitivity than previously described techniques employing TNBS.