A procedure for the rapid, quantitative N-acetylation of amino sugar methyl glycosides.

A rapid and quantitative procedure is described for the re-N-acetylation of amino sugar methyl glycosides prior to their analysis by gas-liquid chromatography. Two equivalents of pyridine are added to acidic methanolysates containing amino sugars, serving both to neutralize the acid and to act as a catalyst for the subsequent N-acetylation reaction with acetic anhydride. The N-acetylation is quantitative and complete within 10 min at ambient temperature. Excess acetic anhydride is destroyed by solvolysis with the methanolic solvent. The procedure has been used effectively for methanolysates containing 0.01–2.0 mg/ml glucosamine. The procedures utilizing ion-exchange columns and insoluble salts are thus circumvented and all reaction byproducts are volatile. The procedure is therefore ideally suited for the simultaneous workup of numerous samples for analytical procedures such as gas-liquid chromatography.     

A method for the quantitative measurement of cell aggregation

It is demonstrated that formation of cellular aggregates in a slowly rotating suspension is accompanied by a decrease in total cell concentration in the top layer of the suspension. Both the average particle size and the initial cell concentration of the homogeneous suspension, are parameters which determine the magnitude of the effect.The method is exemplified by

1. 1. aggregation of HeLa cells after treatment with neuraminidase;

2. 2. agglutination of HeLa cells with concanavalin A;

3. 3. agglutination of human erythrocytes with poly- -lysine;

4. 4. agglutination of human erythrocytes with poly- -lysine following pretreatment with neuraminidase.

     

A proton-relaxation enhancement study of the interaction of manganous ions with phospholipids in aqueous dispersions.

An interaction of dipalmitoylphosphatidylcholine (PC) and phosphatidylserine (PS) with manganous ions has been investigated by measuring the effect of bound manganese upon the longitudinal relaxation rate, 1/T1, of the solvent water protons and evaluating the enhancement factor epsilon b. The observed enhancement values were used to determine the number of interacting sites per polar head group, n, and the values of association constants, KA, of manganese to PC and PS. Changes in epsilon b correlate with structural changes at the interacting site. By increasing the temperature one can see an abrupt decrease in epsilon b within the temperature interval from 40 to 50 degrees C indicating the thermal phase transition of PC as established by calorimetry, fluorescence and high-resolution NMR measurements. That an enhancement of 1/T1 of the solvent-water protons occurs at all is explained by assuming a restricted rotation of the Mn2+-aquo complex in the bound state. In addition we suppose that the rotation of the Mn2+-aquo complex is the mechanism which dominates the relaxation of the water protons in teh bulk solvent when phospholipids are present.     

A quantitative Western Blot method for protein measurement

A radioimmunologic assay method for the quantitation of small amounts of protein in recombinant vaccines at the level of 20-150 ng is evaluated which uses the techniques of SDS-PAGE and electrophoretic protein transfer ("Western Blot'). Known amounts of the protein being determined are included on the same gel as the unknown. After protein blotting, the nitrocellulose membrane is treated with antibody specific for the protein being determined and subsequently with [125I] Protein A. An autoradiogram is produced which corresponds directly to the nitrocellulose blot. It can, therefore, serve as a template to locate the labeled protein which is excised from the blot and measured in a gamma counter. The technique is found especially useful for evaluating cell lysates of recombinant bacteria and yeast for the percentage of the recombinant protein in the total protein mixture.     

A novel method for the quantitative extraction of juvenile hormones from insects including the silverfish, Thermobia domestica

An improved procedure for extraction of juvenile hormones (JH) from large quantities of insects is described. It consists of the following steps. The lyophilised insects are homogenized in cold methanol and, after storage overnight, the debris is removed by filtration and the methanol is evaporated. For the next two steps the residue is dissolved in ether and by adding either methanol in the first step, or acetone in the second step, a precipitate is formed which is removed by filtration. With this method, 2 to 7 times less lipids are extracted than with usual methods involving ether or ethyl acetate, but more than 99.5 per cent of the JH is extracted at least in H. cecropia. The JH activity of the extracts is at least 10 times higher than extracts prepared with ether or ethyl acetate alone.     

Synthesis of determinant disaccharides of O-antigens of Salmonella of serological groups A, B and D1 in the form of glycosides suitable for conversion into artificial antigens via copolymerization

The synthesis of 3-O-(alpha-paratosyl)-, 3-O-(alpha-abequosyl)-, and 3-O-(alpha-tyvelosyl)-alpha-D-mannopyranoses, group-specific disaccharides fragments of the Salmonella O-specific polysaccharides (serological groups A, B, and D1) is described. Disaccharides were prepared in the form of allyl and 2-acrylamidoethyl glycosides suitable for conversion into artificial antigens via copolymerisation.     

A gamma-ray computed tomography scanner for the quantitative measurement of bone density

A special purpose gamma-ray computed tomography scanner has been developed for the precise measurement of bone density in the distal forearm. Details of the scanner hardware and computer analysis technique are given. Suitable phantoms have been used to test the operation of the scanner, which has been used to measure trabecular and cortical bone density with a precision better than 1%.     

A quantitative method for the measurement of cellular guanosine triphosphate pool specific activities

Studies on quantitation of RNA synthesis in eucaryotic cells have frequently used adenosine as the radioactively labeled precursor, largely because of the convenience of the firefly luciferin-luciferase assay in measuring ATP pool specific activity (1,2). This could result in some difficulties if the addition of poly(A) to the 3′ OH end of RNA represents a significant portion of total incorporation, as is the case in sea-urchin embryos (3). In addition, in some cases, the ATP pool may be large enough to prevent the use of adenosine as an effective labeling agent. Hence, a simple and sensitive method for the determination of the specific activity of the other nucleic acid precursor pools would be of value.Although the crystalline luciferase is specific for ATP, extracts of firefly lanterns most commonly used for quantitating ATP (4–9) also exhibit activity with other ribonucleoside triphosphates, adenosine tetraphosphate, ADP, and the deoxyribonucleoside triphosphates. This activity is due to the presence of contaminating enzymes such as nucleoside 5′-diphosphate kinase and adenylate kinase which catalyze the formation of ATP from these nucleotides and trace amounts of ADP, also present in the extracts (10–13). Recently, Manandhar and Van Dyke (14) have reported a procedure for quantitating picomole levels of GTP with a crude extract of firefly lanterns. In the present study, we have adapted their procedure to develop an assay for GTP pool specific activity in Xenopus laevis oocytes microinjected with [8-³H]GTP. Our assay may be extended to the analysis of any nucleoside triphosphate pool, provided that an adequate chromatography system is available for the separation of the extracted nucleotides.     

A quantitative method for the measurement of membrane affinity by polydiacetylene-based colorimetric assay

The measurement of membrane affinity is an important early screening step during drug discovery. However, classical methods for membrane affinity measurement are tedious and difficult to implement in high-throughput screening. This article describes a quantitative method for the measurement of membrane affinity by colorimetric assay based on polydiacetylene (PDA) sensors. Prepared lipid/PDA chromatic vesicles were used to model cell membranes. By measuring the colorimetric response of the chromatic vesicles when drug-membrane interactions occurred, membrane affinity constant K(b) could be calculated using a simple quantitative model. Under optimized preparation conditions, the calculated log(K(b)) values exhibited an in-batch relative standard deviation (RSD) of less than 4% and a between-batch RSD of less than 8% for all three reference compounds. The logarithm of K(b) of the six β-blockers exhibited excellent linear correlation with the logarithm of the liposome/water partition coefficient (K(m)) with R(2)=0.9793. For neutral compounds, the log(K(b)) of n-fatty alcohols correlated with the logarithm of the n-octanol/water partition coefficient (K(oct)) with a linear correlation coefficient R(2)=0.9833. This work provides a simple, convenient, and reproducible method for the rapid measurement of membrane affinity and presents important implications for high-throughput screening.     

A quantitative histochemical procedure for measurement of starch in apple fruits

Measurements of starch (e.g. amyloplasts in stomatal guard cells, sieve elements, root tips or the starch sheath) is often very difficult using most analytical methods. An evaluation was made of interactive computer image analysis of starch measurements in apple fruits. The results obtained indicate that quantitative histochemistry can be an appropriate method to quantify starch. Correlations for starch values between the image analysis system and a colorimetric system were quantified. The thickness of plastic-embedded slices had no influence on the accuracy of the area occupied by image-quantified starch (starch/slice) or on its variance. The magnification of the objective also had no effect on measured starch-occupied areas (starch/slice), but there were big differences in variance. The number of replications required to establish statistically significant differences were calculated.     

A quantitative autoradiographic method for the measurement of local rates of brain protein synthesis

We have developed a new method for measuring local rates of brain protein synthesis in vivo. It combines the intraperitoneal injection of a large dose of low specific activity amino acid with quantitative autoradiography. This method has several advantages: 1) It is ideally suited for young or small animals or where immobilizing an animal is undesirable. 2) The amino acid injection floods amino acid pools so that errors in estimating precursor specific activity, which is especially important in pathological conditions, are minimized. 3) The method provides for the use of a radioautographic internal standard in which valine incorporation is measured directly. Internal standards from experimental animals correct for tissue protein content and self-absorption of radiation in tissue sections which could vary under experimental conditions.A preliminary report of this work was presented at the 11th annual meeting of the Society for Neuroscience, Los Angeles, California, October 18–23, 1981.     

A microassay for the quantitative measurement of egg yolk-reactive enzyme activity produced byPseudomonas cepacia

A newly developed microassay offers a sensitive method for quantitating egg yolk reactivity in culture supernatants and samples prepared during enzyme purification. Equal volumes of supernatant, saline, egg yolk suspended in saline, and buffer were incubated in microtiter wells at 37°C, and the resulting turbidity was measured quantitatively with an ELISA reader at 410 nm. The microassay was used to screen culture supernatants from nine clinical isolates ofPseudomonas cepacia, and the results were compared with those obtained when the isolates were screened on egg yolk agar. The microassay was also used to detect egg yolk reactivity in ammonium sulfate-precipitated fractions of the culture supernatant of one strain, Pc224c, and to determine which fraction of egg yolk contained the substrate for the activity.     

Individual differences in plasma ALT, AST and GGT: contributions of genetic and environmental factors, including alcohol consumption

The causes of individuality of the plasma enzymes alanine aminotransferase (ALT; EC 2.6.1.2), aspartate aminotransferase (AST; EC 2.6.1.1) and gamma-glutamyl transferase (GGT; EC 2.3.2.2) were investigated in a study of 206 pairs of twins. Between-person variance was greater in men than women, while within-person variation was similar in both sexes. Plasma ALT and AST levels were affected by genetic factors, while GGT was affected by some environmental factor shared by co-twins. In the men, alcohol intake had a significant but small effect on all three enzyme levels, and since alcohol consumption was highly heritable, this appeared as a genetic influence on enzyme activities. The major factors involved in the observed correlations between these enzymes were a non-shared environmental factor other than alcohol affecting ALT, AST and GGT, and a genetic factor affecting only ALT and AST.