THE POWER OF THE " A" -" NOT A" METHOD

The " A" - " Not A" method is a rating method with two categories. It is often treated as a discrimination method. Unlike forced choice procedures, the Thurstonian model for this method involves a choice criterion. In statistical tests, it is treated as a comparison of two proportions. In this paper, the power for hypothesis tests involving the monadic and replicated monadic " A" - " Not A" method is discussed. The power functions and the sample sizes needed for 80% power are given based on Thurstone's δ. Designs with equal and unequal allocations for A and A (Not A) samples are considered. The power of the method is also compared with that of four forced choice methods under the assumption that the perceptual variance is identical among methods. The comparison shows that, in general, the power for the five methods ranks from high to low: the 3-AFC, 2-AFC, " A" - " Not A", triangular and duo-trio. The comparison also shows that, based on the same number of panelists and/or the same sample size for the A and A⁻ samples for the methods, if the panelists are not too discrepant and the choice criterion in the " A" - " Not A" method is not too strict or too lax, the power of the " A" - " Not A" method is very close to that of the 2-AFC method.     

The office treatment of tattoos: a simple and effective method

A simplified method for the treatment of tattoos is described. The method is based on an older method called the "French method" known to tattoo artists. The treatment consists of superficial dermabrasion of the skin followed by application of a tannic acid solution which is then "rubbed" into the skin by the dermabrasion wheel. The treatment site is then "painted" with a silver nitrate stick. A heavy eschar forms which separates in approximately 2 to 3 weeks. The method incorporates the use of equipment that is present in the usual plastic surgical office. The method has produced excellent improvement in tattoos, with obliteration of the tattoo in the majority of cases. This report covers 85 patients with 207 tattoos treated over an 11-year period. The method is presented as a treatment of tattoos that are too large for simple excision. Details of the treatment are presented.     

Determination of the concentration of ligand-specific molecules, found in mixtures with ligand-nonspecific molecules

A new method, which allows determination of the concentration of ligand-specific molecules in a mixture of these molecules with biochemically similar but ligand-unspecific molecules, is suggested. The method is based on a partial exhaustion of the mixture on a column with immobilized ligand and determination of the part of ligand-specific molecules presented in exhausted mixture. The concentration of monoclonal antibodies specific to bovine serum albumin in a commercial "Sigma" preparation and concentration of polyreactive immunoglobulins in a commercial "Sigma" preparation of bovine immunoglobulins were determined by suggested method.     

Spectrophotometric determination of the quantity of protein in standard biological suspensions

A combined method for spectrophotometric measurement of protein amount for conventionally isolated biological suspensions is suggested. The method is based on the comparison of data on the optical density of a suspension at 280-290 nm with the result of a single colorimetric determination. For the membranes of sarcoplasmic reticulum and mitochondria it has been shown that the combined spectrophotometric method gives possibility to shorten the time of measurement, has high specificity and sensitivity and compares favourably with usual colorimetric methods in relation to accuracy. A calibration chart was obtained for conventionally isolated "Wistar" rat liver mitochondria.     

Isolation of Pinus radiata Genomic DNA Suitable for RAPD Analysis

A protocol is presented for Pinus radiata genomic DNA isolation based on an alkyltrimethyl-ammonium bromide (CTAB) method described for other woody species. The method involves mortar grinding of tissue, a modified CTAB extraction employing high salt concentrations and polyvinyl pyrrolidone, a RNase A treatment and successive isoamyl alco- hol-chloroform extractions. The yield was approximately 15 g DNA per 100 mg of initial fresh plant material. The genomic DNA obtained by this method was suitable to be used in simple sequence repeat and random-amplified-polymorphic DNA reactions. This extraction method would allow the molecular analysis of shoots from different clones within P. radiata families.     

Identification of RNA-recognizing modules on the surface of ribosomal proteins

Properties of specific interaction between ribosomal proteins and ribosomal RNAs were analyzed and a method for determination of "recognizing modules" on the protein surface was proposed. The method is based on the search of protein atoms making conserved H-bonds with RNA and forming an invariant spatial structure in homologous rRNA-protein complexes and in the isolated protein. A potential of the method is demonstrated on the determination of the recognizing modules on the surfaces of ribosomal proteins S8, S15 and L5.     

Preparation of samples of biological fluids for electron spin resonance (ESR) examination

The ESR (Electron Spin Resonance) Technique, is usually used in the study of Free Radicals and Metal - Proteins, in biological fluids, but the most apt method for the preparation of the sample has still to be defined. In this work the A.A. describe and analyse the negative and positive aspects of several methods. The results of the E.S.R. spectra, obtained by means of lyophilization, that is the most used method in literature, have been compared with those obtained by means of concentration of some biological fluid (serum and synovial fluid). The A.A. demonstrated that the lyophilization does not permit the reproducibility of the spectra, for different spectra are obtained from the same lyophilized serum and synovial fluid samples. A high reproducibility of the spectra is instead obtained by the concentration of the sample means of the "collodion bag" or "Aquacide" methods. The A.A. are convinced that for on exact investigation of the E.S.R. Signals, only the concentration method is useful, and not the lyophilization method as reported in literature.     

Effect of adenosine protonation on its interaction with polyuridylic acid

The effect of adenosine protonation on complex formation between poly(U) and adenosine has been studied by UV spectroscopy, titration and equilibrium dialysis techniques. A method has been developed to estimate the "misincorporation" of ionized monomer molecules into a polynucleotide--monomer complex. The method is based on combining the titration and binding data. Using this method it is shown that protonated adenosine interacts to some extent with poly(U) in the course of A.2 poly(U) dissociation at acidic pH. Qualitative differences between the effects of ionization of the polymer and monomer components on polynucleotide--monomer interaction are discussed.     

A simple quantitative method of estimation of cell-intactness based on ethidium bromide fluorescence

A quantitative method based on fluorescence generated by the binding of ethidium bromide (EB) to DNA has been developed for estimation of the intactness of the plasma membrane of a mammalian cell type (goat epididymal spermatozoon). The method consists of mixing of sperm preparations with EB in a modified Ringer's solution followed by immediate measurement of fluorescence intensity at 365-580 nm (excitation-emission). The data were corrected for non-specific values of fluorescence due to intact cells only. The percentage of damaged cells in a sperm population was calculated by comparing the corrected fluorescence values of the cell preparations with those of the sonicated cells. The values of sperm intactness obtained by this method (99.5 +/- 0.3) compared well with those obtained by the widely used "marker enzyme" method (97 +/- 0.8) based on estimation of lactic dehydrogenase in the extracellular medium. The validity of this method has been confirmed by using cells of defined intactness i.e. preparations of vigorously forward-motile spermatozoa that showed nearly 100% intactness. The method can detect as low as 0.5% "leaky" or damaged cells in a cell preparation. The "EB-fluorescence" method is simpler and more rapid and reliable than the conventional "marker enzyme" method for estimation of cellular intactness.     

Use of protein A in the immunosorbent analysis of antibodies to the tick-borne encephalitis virus

The results of the studies made with a view to developing the method for the determination of specific antibodies to the antigen of tick-borne encephalitis virus in human blood serum and liquor are presented. The method is based on the capacity of Staphylococcus aureus protein A to bind with Fc-region of immunoglobulins, which makes it possible to use this protein as the "second" system of antibodies. The conditions for the sorption of the antigen on polystyrene test tubes and for binding 125I-or horse radish peroxidase-labeled protein A preparations with antibodies have been determined, and the method has been approved in tests made on sera and liquor obtained from donors and tick-borne encephalitis patients.     

A method to determine the end point of decalcification of hard tissue and bone

A method for decalcification end point determination of mineralized tissue is described. The calcium content of the decalcification solution was determined colorimetrically with a "continuous automatic analyzer" with a high degree of accuracy. The end point method used has been tested on two decalcification methods, 5% nitric acid with or without ultrasonic treatment. The results suggest it is possible to quantitate the decalcification process.     

3D structure of bovine pancreatic ribonuclease A in aqueous solution: An approach to tertiary structure determination from a small basis of1H NMR NOE correlations

Summary A method is proposed to generate initial structures in cases where the distance geometry method may fail, such as when the set of¹H NMR NOE-based distance constraints is small in relation to the size of the protein. The method introduces an initial correlation between the and backbone angles (based on empirical observations) which is relaxed in later stages of the calculation. The obtained initial structures are refined by well-established methods of energy minimization and restrained molecular dynamics. The method is applied to determine the solution structure of Ribonuclease A (124 residues) from a NOE basis consisting of 467 NOE cross-correlations (97 intra-residue, 206 sequential, 23 medium-range and 141 long-range) obtained at 360 MHz. The global shape and backbone overall fold of the eight final refined structures are close to those shown by the crystal structure. A meaningful difference in the positioning of the catalytically important His¹¹⁹ side chain in the solution and crystal structures has been detected.     

The "block" approach in x-ray diffusion dispersion as a method of studying the packing of secondary structure elements in globular proteins

To calculate large angle scattering intensity of biopolymers in solution a "block" method of modeling the globular proteins structures is suggested: conformation of protein alpha-helical and beta-structural parts are described in detail, whereas the remaining part of the macromolecule is substituted by a continuum with homogeneous electron density. A comparison of large-angle scattering curves on the basis of the "block" method and the exact structure was made for a number of alpha-helical and beta-structural globular proteins. It is shown that there is a qualitative agreement between the "exact" and model curves.     

Isolation of DNA from single microsurgically excised bands of polytene chromosomes of Chironomus

A method is described for excising by a glass knife single bands of isolated polytene chromosomes of the salivary glands of Chironomus tentans larvae. DNA strands were isolated from cut-out bands and their contour lengths were determined on electron micrographs. The mean contour length of DNA strands isolated from the double band I-8A was about twice that of the single band I-11B, namely 63 versus 34 m. The described method may be applicable for molecular studies on single bands (e.g., by DNA cloning).     

Automatic analysis of time-sequence parameters in unit cell activity

A method is presented which allows to automatically evaluate, by the aid of a small computer, parameters concerning the way a sequence of spikes is organized in time. This method stands on few simple assumptions: Interspike Time Intervals (ITIs) and/or clusters of ITIs that are either "too large" or "too short" so as to be unlikely at 0.05 level on the basis of a Poissonian firing hypothesis are selected and assumed as "Low Firing Periods" (LFP) and "High Firing Periods" (HFP), respectively. General information is obtained about both LFPs and HFPs. Results on real cases are in agreement with previous studies on unit cell activity.     

Improvement of DNA quantitation following proteolytic extraction

A reliable and sensitive method for quantitation of deoxyribonucleic acid (DNA) is described herein. It is based on a proteinase K digestion step followed by a fluorometric assay with 3,5-diaminobenzoic acid(Kissane and Robins, 1958). The method proved to be applicable to fresh liver and to hepatocytes either in suspension or in culture when attached to a collagen substratum. A linear relationship between biological sample size and fluorometric response was observed only if the assay was preceded by proteolytic extraction. Moreover, the method resulted in DNA values per fresh liver weight that were about 100% higher than values reported earlier. The data show that proteolysis results in a more exhaustive extraction of DNA from tissues and a better separation of extracted DNA from other tissue constituents interfering with the assay.     

Quantitative measurement of starch in very small amounts of leaf tissue

Summary A specific enzyme method is described for the routine estimation of starch in small quantities (10–30 mg) of dried leaf tissue. A -glucanase-free preparation of amyloglucosidase is employed to hydrolyse starch to glucose; this is subsequently estimated by the glucose oxidase technique. The method gives result which agree closely with those obtained by a specific iodine-precipitation method.     

The use of β-galactosidase as a tracer in immunocytochemistry

Summary A new immunocytochemical method using -galactosidase as a tracer is described. The positive staining appears blue on an unstained background. The present method has the high sensitivity and specificity of the immunoperoxidase method and appears to be a practical alternative. The substrate has no carcinogenic activity. Staining is permanent and the sections can be dehydrated and mounted in synthetic media. Enzyme and substrate solutions are stable for several months.