Assays Warren

The activity of several lytic enzymes, including sialidase, is necessary for fertilization. While sperm are in the seminal plasma, inhibitors of these enzymes are present in the decapacitation factor (DF). Sialidase was found to be a component of sperm acrosomes by Srivastava et aL (1970). They found sialidase activity and a sialidase-like factor which rendered bound sialic acid reactive in the thiobarbituric acid assay (Warren, 1959) but did not release it from the molecule and, upon purification, appeared to be converted to sialidase. An inhibitor of the sialidase-like factor was found in a partially purified DF preparation. The DF is lost during capacitation, which can be broadly defined to include the changes sperm undergo to enable them to penetrate the vitelline membrane of the egg. Gould et aL (1971) have proposed that the role of sialidase in fertilization is to alter the tertiary and quaternary structure of sialoglycoprotein present in the zona pellucida to cause the zona pellucida to be less penetrable by spermatozoa and serve to reduce or block polyspermy. 

G.A. Umbuzeiro, H. Freeman, S.H. Warren, F. Kummrow and L.D. Claxton, Mutagenicity evaluation of the commercial product Cl Disperse Blue 291 using different protocols of the Salmonella assay. Food Chem. Toxicol. 43 (2005) 49-56. 

Warren L (1959) The thiobarbituric acid assay of sialic acids. J Biol Chem 234 1971-1975... 

Felix JP, Williams BS, Priest BT, Brochu RM, Dick IE, Warren VA, Yan L, Slaughter RS, Kaczorowski GJ, Smith MM, Garcia ML (2004) Assay Drug Dev Technol 2 260... 

Moisture. In NIR, as in the MIR, water has the strongest absorption of light. It stands to reason that moisture in solid products would be an important assay. For instance, Warren et al. used NIR to determine the amount of water in glycerides. Transmission spectra of standard... 

E364 Mauck, J.C., Smith-Lewis, M.J. and Warren, H.C, (1987). A coated multilayer colorimetric assay for serum iron. Ann. Clin. Biochem. 24, Suppl. 2, 209. 

E579 Warren, P. and Lott, J.A. (1989). Evaluation of Kodak slides for cerebrospinal fluid protein assays. Clin. Chem. 35, 1090-1091, Abstr. 118. 

ENl 16 Mauck, J., Warren, H. and LaTart, D. (1992). Development of a thin-film colorimetric assay for serum leucine aminopeptidase. Clin. Chem. 38, 985-986, Abstr. 217. 

Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren J, Bokesch H, Kenney S Boyd MR (1990) New colorimetric assay of anticancer drug screening. Journal of the National Cancer Institute 82 1107-1113. 

Whicher JT, Warren C, Chambers RE. Immunochemical assays for immunoglobulins. Ann CUn Biochem 1984 21 78-91. 

Evenson and Warren developed an assay for theophylline using microparticulate silica gel columns and a mobile phase of water-saturated chloroform - heptane - acetic acid (300 200 0.4) to which 6% ethanol was added. Heptane was necessary to achieve a separation of theobromine... 

Fig. 8. EPR assay for the reconstituted complex [P700 Ao A, FeS-X] FeS-A/B PsaD PsaE containing either the PsaC Cys-14->Asp (A) or the PsaC Cys-51->Asp mutant protein (B). The experimental protocol is described at the top ofthe figure all spectra were measured at 15 K. Spectra (a) are for dark-frozen samples and (b) for the same sample under illumination at 15 K. Spectra (c) are the light-minus-dark difference spectra, i.e.. (b)-(a), presented at an additional gain of 3.65X. Spectra

Seventy-six gene profile Prognostic for lymph node negative irrespective of HR status Oligonucleotide microarray Good prognosis versus Poor prognosis Fresh/frozen Rotterdam Assay-in development by Veridex, Warren, NJ)... 

Warren, D.J., Bjemer, J., Paus, E., Bprmer, O.P., and Nustad, K. (2005) Use of an in vivo biotinylated single chain antibody as capture reagent in an immunometric assay to decrease the incidence of interference from heterophilic antibodies. Clinical Chemistry, 51, 830 838. 

Sheppard, J., N.D. Warren, and P.D. Case The effect of cigarette design variables on assays of interest to the tobacco industry 2) Prediction of smoke and Hoffmann analytes using two different modehng methods CORESTA Joint Study Group Meeting, Startford-on-Avon, U.K. (2005) Paper No. SSPT 02. 

Claxton LD, Woodall GM (2007) A review of the mutagenicity and rodent carcinogenicity of ambient air. Mutat Res Rev Mutat Res 636 36-94 Claxton LD, Matthews PP, Warren SH (2004) The genotoxicity of ambient outdoor air, a review Salmonella mutagenicity. Mutat Res Rev Mutat Res 567 347-399 Clemons JH, Allan LM, Marvin CH, Wu Z, McCarry BE, Bryant DW, Zacharewski TR (1998) Evidence of estrogen- and TCDD-like activities in crude and fractionated extracts of PMIO air particulate material using in vitro gene expression assays. Environ Sci Technol 32 1853-1860... 

Fig. 2. Inactivation of estradiol 17/3-dehydrogenase by 16a-bromoacetoxyestra-diol 3-methyl ether and protection against enzyme inactivation by cofactor or substrate. Aliquots were assayed each point is the mean of four determinations. Enzyme (1.4 nmoles) was preincubated with 16o -bromoacetoxyestradiol 3-methyl ether (0.21 /tmole) in 15 ml of buffer A, 25°, pH 7.0 containing 20% ethanol, (O) 16a-bromo-acetoxyestradiol 3-methyl ether (051 nmole) and NADH (0.42 /tmole), (A) 16 -bromoacetoxyestradiol 3-methyl ether (051 /amole) and NADPH (0.42 /tmole) ( ) lOa-bromoacetoxyestradiol 3-methyl ether (051 /imole) and estradiol (051 /imole) ( ) and control or with bromoacetic acid (0.42 mole) (A). From C. C. Chin and J. C. Warren, J. Biol. Chem. 250, 7682 (1975).

Calf endometrium cytosols were prepared in 2.2 Af sucrose, 3 mM MgClt. Preincubations of cytosol were carried out at 0° for 30 min with and without steroids (10 nAf) and incubated with nuclei at 25° for 30 min. Nuclei were separated subsequently and assayed for RNA synthesis activity. From N. R. Kanamar-lapudi and J. C. Warren, J. Biol. Chem. 260, 6484 (1975). 

Assay by Reference Standard. It has been realized that the dependence of the turbidimetric assay on a substrate with suitable properties limits the general usefulness of the method to some extent. This shortcoming has been eliminated by the use of a reference standard for hyaluronidase as used by Warren et al. (192) and Alburn and Whitley (1). The enzyme standard is assayed simultaneously with the unknown samples, and the unknown activities are calculated from the turbidity reduction curve of... 

Warren GR (1982) Detection of genetically toxic metals by microliter microbial DNA repair assay. In Waters MD, Sandu SS, Huisingh JL, Claxton LD, Nes-now S (eds) Short term bioassays in the analysis of complex mixtures II. PB 82-233172, U.S. Environmental Protection Agency, Research Triangle Park, NC, pp 101-118. 

The Periodic AcidIThiobarbituric Acid Assay a) The Warren and Aminoff Methods... 

The periodic acid/thiobarbituric acid assay has been automated by several authors for use with autoanalysers. For instance, Kendall (1968) and Fidgen (1973) employed the conditions of the Aminoff (1961) assay including organic solvent extraction. This assay was also used by Gerbaut er al. (1973), but without extraction, for measurement of sialic acid in serum. Adaptation of the Warren assay to the autoanalyser was reported by Delmotte (1968), who also dispensed with extraction of the chromophore. This method does not correct for 2-deoxyribose contamination (Delmotte 1968) and has subsequently been adapted to analyze both sialic acid and 2-deoxyribose in the same samples from blood and tissues (Engen et al. 1974) by measuring the optical density at 550 and 530 nm. 

When L-valine-l- C was added to suspensions of washed mycehum of the Cephalosporium sp. the specific radioactivity of extraceUular penicilhn N reached a maximum within a few minutes and then began to fall. The specific radioactivity of cephalosporin C continued to increase for some time while that of penicilhn N was fading, but the apparent specific radioactivities of the two antibiotics did not follow a course which could be interpreted in terms of a product-precursor relationship between them. However, some uncertainty attended the quantitative aspects of these experiments, in which the amounts of antibiotics were deduced from biological assay and their radioactivities from counts on paper after electrophoresis and chromatography (Warren et al., I967). Whether cephalosporin C is formed from penicilhn N is thus still an open question, although it would be difficult to reconcile this biosynthetic pathway with the apparent abihty of D-valine to depress selectively the production of penicilhn N. 

To date, various assay methods for sialidase activity have been developed. In principle, the hydrolytic reaction catalyzed by sialidase can be assayed by quantitating the released sialic acid or aglycone. The thiobarbituric acid procedure described by Warren (1959) or its modification by Aminoff (1961) is used frequently for the colorimetric determination of released, free sialic acid. 

Sialic acids were reported to occur in all species of vertebrates and in certain invertebrates as well (Warren, 1963a), NANA is present in some strains of E, coli and other bacteria as a homopolymer, of which colominic acid is one example (Barry and Goebel, 1957 Barry et al., 1962 Watson et al., 1958 Liu et aL, 1971). However, it is lacking in other bacteria (Barry, 1959 Barry et aL, 1%3 Jann, 1969). Earlier claims for the presence of sialic acid in plant extracts have been disputed (Cabezas and Feo, 1969) since the positive test with thiobarbituric acid could not be duplicated by other colorimetric assays. Gielen (1%8) demonstrated that the color-producing substances in certain plant hydrolysates were 2-keto-3-deoxyaldonic acids rather than sialic acid. To date there has been no convincing evidence for the presence of sialic acid in plants. 

The other widely employed colorimetric assay is the thiobarbituric acid procedure which is useful for free but not bound sialic acid. Proposed independently by Warren (1959) and Aminoff (1959), the method is based on periodate oxidation of sialic acid to )8-formylpyruvic acid (Figure 11) which then reacts with two moles of 2-thiobarbituric acid to form a chromophore (XLIX) with absorption maximum at 549 nm. The identity of this chromophore was established through synthesis... 

A scaled-down version of the Warren method was developed that can be used to assay as little as 25 ng of NANA (Hahn et aL, 1974). A number of automated procedures based on periodate-thiobarbituric acid have been described (Kendal, 1968 Delmotte, 1968 Fidgen, 1973 Gerbant et aL, 1973). Additional spectrophotometric techniques have employed the sulfo-phospho-vanillin reaction (Saifer and Feldman, 1971) and 1,10-phenanthroline (Dimitrov, 1973). The direct Ehrlich reaction, utilizing dimethylaminobenzaldehyde, played a key role in the early studies of sialic acid chemistry and analysis (Gottschalk, 1960) and still finds occasional use although its sensitivity is considerably below that of resorcinol and thiobarbituric acid (Werner and Odin, 1952 Onodera et aL, 1965). In comparing this method with others, Onodera et al. (1965) concluded that the possibility of false chromophores requires at least two colorimetric methods for reliable estimate of sialic acid in biological materials. 

Warren, L., 1963, Thiobarbituric acid assay of sialic acid. Methods Enzymol. 6 463. Warren, L., 1964, N-Glycolyl-8-O-methylneuraminic acid. A new form of sialic acid in the si3T ish Asterias forbesi, Biochim. Biophys. Acta 83 129. 

Warren, L., 1959, The thiobarbitunic acid assay of sialic acid, 7. Biol. Chem. 234 1971. 

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