A new pair for inter- and intra-molecular FRET measurement

Fluorescence resonance energy transfer between mutant green fluorescent proteins provides powerful means to monitor in vivo protein-protein proximity and intracellular signaling. However, the current widely applied FRET pair of this class (CFP/YFP) requires excitation by expensive UV lasers, thereby hindering FRET imaging on many confocal microscopes. Further challenges arise from the large spectral overlap of CFP/YFP emission. Another FRET pair GFP/DsRed could obviate such limitations. However, the use of DsRed as a FRET acceptor is hampered by several critical problems, including a slow and incomplete maturation and obligate tetramerization. A tandem dimer mutant of DsRed (TDimer2) has similar spectral properties as those of DsRed. The rapid maturation and non-oligomerization make TDimer2 a promising substitute for DsRed in FRET experiments. Here, we have explored the possibility of using TDimer2 as a FRET acceptor for the donor EGFP. FRET was demonstrated between the EGFP-TDimer2 chimeric fusion protein. By substituting CFP/YFP in the Ca2+-sensor cameleon with EGFP/TDimer2, dynamic changes in cytosolic free Ca2+ concentrations were observed with 488nm excitation under conventional wide-field microscopy. The EGFP/TDimer2 pair was further successfully employed to monitor inter-molecular interaction between Syntaxin and SNAP25. These results reveal EGFP/TDimer2 as a promising FRET pair in monitoring intra-molecular conformation change as well as inter-molecular interaction.     

A new method for protease activity measurement.

A new method for protease activity measurement is described. In the presence of excess leucine aminopeptidase from Aspergillus japonica, action of protease on succinyl-casein results in the production of l-amino acids and their amino acids are simultaneously determined by l-amino acid oxidase-peroxidase system. Our proposed method is less time consuming and has a much higher sensitivity than the casein-Folin method. The present method is suggested to be suitable for the assay of neutral or alkaline proteases from animals and microorganisms.     

A new system for automatic measurement of Permanganate Time

Permanganate Time (PT) is a standard quality control test for oxidisable impurities in products such as ethanol, acetic acid, acetic anhydride, acetone, methanol, pyridine, propionic acid and tricresyl phosphate. The test relies on visual assessment of the change in pink colouration over a period of time of a sample of product to which has been added a standard solution of potassium permanganate. Typically, this time period is between 30 min and 2 h. The standard test, therefore, is very time-consuming as well as being highly susceptible to operator error and bias. An automated measurement system has been developed that employs a photodiode array spectrophotometer to monitor the colour of the mixture. Pre-processing of the spectrum eliminates any interference and provides accurate estimates of the PT, even in the presence of a heavy precipitate. The method by which the software determines the test result can be tailored by the user to suit particular products. Any number of such user configurations can be saved for future use.     

Sublingual mucosa: A new vaccination route for systemic and mucosal immunity

Needle-free vaccine delivery has become a global priority, both to eliminate the risk of improper and unsafe needle use and to simplify vaccination procedures. In pursuit of greater ease of vaccination, a number of needle-free delivery routes have been explored, with mucosal routes being perhaps the most prominent. Since the vaccine administration route significantly affects immune responses, numerous researchers are attempting to develop alternative vaccine delivery methods including a mucosal route. My group's recent studies demonstrate the potential of the sublingual (s.l.) route for delivering vaccines capable of inducing mucosal as well as systemic immune responses. Sublingual administration conferred effective protection against a lethal challenge with influenza virus (H1N1) or genital papillomavirus. Moreover, CCR7-CCL19/CCL21-regulated dendritic cells are responsible for activation of T and B cells following s.l. administration. This review highlights current knowledge about the safety and effectiveness of s.l. vaccination and describes how s.l. vaccination can induce both systemic and mucosal immunity.     

A new substrate for the measurement of N-acetyltransferase activity

A sensitive colorimetric method for the measurement of N-acetyltransferase (NAT) is described. It is based on the high rate of acetylation of 2-(p-aminobenzamido)pyridine by the liver enzyme and the lack of it by the blood NAT. A linear relationship was found between enzyme concentration and reaction rate. The reaction rate was also proportional to the substrate concentration. Inhibition of the reaction was observed at high substrate concentrations. The NAT levels in the liver and kidney of rat, rabbit, mouse and man were measured using this procedure. The tissues of dog failed to acetylate this substrate. The method is applicable to kinetic studies such as the analysis of inhibition reactions with o-phenanthroline and p-chloromercuribenzoate.     

A new instrument for the measurement of air ions

The intensive discussion of the possible impact of atmospheric electricity in theInternational Journal of Biometeorology has motivated many physicians and scientists to include the respective measurements along with the assessment of the traditional parameters. The parameters most often required are: air ions, air-to-ground currents and the electrical earth potential. This paper is primarily concerned with a new generation of instruments to study such electrometeorological parameters, particularly air ionization. This paper describes the key data and functions of the Ion-Meter PN-2001 the first result of a mid-term development plan; the Meteoline uP-2000. The latter is a group of five new instruments for the study of electro-atmospheric parameters.     

A new sensitive assay for the measurement of hydroperoxides

The enzyme glutathione (GSH) peroxidase can be used to measure hydroperoxides quantitatively, easily, and specifically. A timed reaction of GSH peroxidase, coupled with the oxidation of NADPH by GSH reductase, allows a direct spectrophotometric measurement of hydroperoxide. Addition of catalase prior to the addition of GSH peroxidase permits the distinction between hydrogen peroxide and organic hydroperoxides. The solvents that can be used with the assay include methanol, ethanol, water, and aqueous solutions of detergents such as Brij 35, Triton X-100, and cetyl trimethyl ammonium bromide. The utility of the method is demonstrated by the measurement of hydrogen peroxide and organic hydroperoxides formed upon ozonolysis of an unsaturated fatty acid.     

A new animal model of Bordetella pertussis infection and immunity

Bordetella pertussis is a strictly human pathogen. Experimental infection of other animals can occur with large inoculating doses; rat, mice and primate models have been used to study pathogenesis and immunity. Recently, it was shown that newborn piglets are susceptible to B. pertussis. Lung pathophysiology of infected piglets was similar to that of human infants that develop bronchopneumonia. Piglets and infants are anatomically similar and maternal antibodies are transferred and secreted by a similar mechanism. This model could be valuable for studying the roles of passively and actively acquired immunity against B. pertussis.     

On the ability of neural networks to perform generalization by induction

The ability of neural networks to perform generalization by induction is the ability to learn an algorithm without the benefit of complete information about it. We consider the properties of networks and algorithms that determine the efficiency of generalization. These properties are described in quantitative terms. The most effective generalization is shown to be achieved by networks with the least admissible capacity. General conclusions are illustrated by computer simulations for a three-layered neural network. We draw a quantitative comparison between the general equations and specific results reported here and elsewhere.     

CARNOY: A new digital measurement tool for palynology

Quantitative data play an important role in palynological research. With the advent of digital imaging in light and electron microscopy, palynologists now have the opportunity to perform measurements faster and more precisely than ever before. Several image analysis software packages already exist for these tasks, but they are often expensive, difficult to use or not adapted to the specific needs of palynologists. After studying the daily workflow of a palynologist, we designed CARNOY, an image analysis application written from the ground up for use in palynology and morphology. CARNOY offers an easy-to-use interface and several features to make measuring easier and faster. The program can export measurements to almost every other software package for further analysis and is available for free on the Internet.     

A new analysis for membrane noise. The integral spectrum.

A new method of random data analysis has been developed with special implications for membrane noise. The integral spectrometer uses overlapping broad-band filters of simple design, whose bandwidth increases linearly with center frequency. A random two-state process, which has a Lorentzian-shaped spectral density, results in an integral spectrum whose maximum value occurs at the mean frequency of the events, and which is symmetric about that frequency on a semilog plot. 1/f noise is flat and does not distort the symmetry on the frequency axis. The integral spectrum exchanges resolution on the frequency axis for accuracy in the amplitude. The expected statistical error in amplitude has been calculated for three types of membrane noise assuming finite data. The integral spectrum compares favorably with conventional spectral densities and may be a reasonable alternative for random data analysis.     

A new method for the measurement of protein turnover.

A new technique for the determination of rate constants of protein degradation is described. By using the method, half-lives of total soluble protein of Lemna minor during growth on full culture medium and distilled water were measured. The method involves incubating Lemna on a growth medium containing 3H2O. After a short exposure (20 min) to 3H-labelled culture medium, 3H was found in soluble amino acids, especially aspartate, glutamate, glutamine and alanine. After transfer to a 3H-free medium for 30 min, 80% of the 3H originally present in soluble amino acids was lost. These results suggest that 3H enters and leaves amino acids at the alpha-carbon atom, a conclusion supported by the observed labelling of glutamates. The exchange of H and 3H on the alpha-carbon atom is catalysed by transaminases and the speed of this exchange ensures that when the 3H2O is removed, the 3H in free amino acids is rapidly lost, thereby eliminating problems connected with metabolic pools and recycling. After an exposure of 20 min to 3H-labelled medium all protein amino acids, except for arginine, were found to be radioactive. The loss of radioactivity from protein amino acids was used to measure protein degradation.     

A new micromethod for routine measurement of serum LDL-apolipoprotein B

A new method for low density lipoprotein (LDL) (d 1.019-1.063 g/ml)-apolipoprotein B (apoB) determination has been developed, based on the fact that very low density and intermediate density lipoproteins (VLDL and IDL) contain apolipoprotein C-I (apoC-I), whereas this apolipoprotein is apparently absent in LDL. VLDL and IDL were quantitatively precipitated with a monospecific anti-apoC-I antibody whereafter LDL-apoB in the supernatant was quantitated by Laurell rocket electrophoresis. Over a wide range of cholesterol and triglyceride values there was a linear correlation with LDL-apoB values measured after ultracentrifugation. The method would be useful for routine measurements, especially in children, since only 25 microliter of serum is required, and for making the diagnosis of hyperapobetalipoproteinemia, which at present is complicated.     

A new method for fluorometric measurement of proline in grapes and wines

A method for the measurement of free proline in grapes and wines containing other amino acids is presented. The method included the removal of phenols and tryptophan by the pre-treatment of samples with activated carbon, the oxidation of proline to the corresponding primary amine with sodium hypochlorite, destruction of other amino acids by oxidation at pH 5.3 using a combination of hydrogen peroxide, ferric chloride, and sodium hypochlorite, and the formation of afluorescent product from the oxidized proline with o-phthalaldehyde in the presence of 2-mercaptoethanol and Brij 35 at pH 5.3. The proline in grapes and wines measured by this method agreed well with the values obtained by the method using an automatic amino acid analyzer with ninhydrin.     

A new instrument for the measurement of forces on beds and seats

Knowledge of the interaction of forces between persons and the bed in which they lie or the seat on which they are sitting, provides an insight into the loading of their muscles, bones and soft tissue. To determine the total forces on the body-supporting surfaces (backrest, seat pan, foot rest) resolved in components perpendicular and parallel to these surfaces a new instrument has been developed, with which the forces perpendicular and parallel to three different freely adjustable body-supporting surfaces can be registered. During the first measurements the forces on a bed were measured when a person sits in a bed with the backrest at an angle of 45° to the horizontal and the mattress horizontal. The measurements on a healthy population (mean mass = 77 kg, sd = 11 kg) showed an accuracy of ± 10 N. In this position the mean shear force on the seat pan was 97 N.