Antibody-targeted photolysis: in vitro immunological, photophysical, and cytotoxic properties of monoclonal antibody-dextran-Sn(IV) chlorin e6 immunoconjugates.

A set of anti-melanoma immunoconjugates were prepared which contained chlorin e6: antibody molar ratios of 18.9:1, 11.2:1, 6.8:1, and 1.7:1. All immunoconjugates retained antigen binding activity regardless of the chromophore:antibody substitution ratio that was attained. In contrast, the ground-state absorption spectra of the immunoconjugates showed features which appeared to be dependent on the chromophore:antibody molar ratio. In addition, the quantum yield of singlet oxygen generated by the conjugated chromophores was observed to be significantly less than that observed with the unbound dye. Time-resolved absorbance spectroscopy of the chromophore excited triplet state indicated that the loss of singlet oxygen quantum yield resulted from diminished chromophore triplet yield. Analysis of data obtained from in vitro photolysis of target melanoma cells, in combination with that obtained from the immunochemical and photochemical studies, indicates that the observed immunoconjugate phototoxicity can be reasonably quantitatively represented by (1) the ability of the immunoconjugate to bind SK-MEL-2 cell surface antigen, (2) the amount of chromophore localized at the target cells by immunoconjugate binding, (3) the delivered dose of light at 634 nm, and (4) the singlet oxygen quantum yield of the antibody-bound photosensitizer. Though these data argue strongly for photolysis by the cumulative dosage of singlet oxygen at the cell membrane, nonetheless, the concurrent photoinduced release of other cytotoxic agents should not be ruled out.     

Oxidation of glycated proteins: age-dependent accumulation of N epsilon-(carboxymethyl)lysine in lens proteins

N epsilon-(Carboxymethyl)lysine (CML) has been identified as a product of oxidation of fructoselysine (FL) in glycated (nonenzymatically glycosylated) proteins in vitro and has also been detected in human tissues and urine [Ahmed et al. (1986) J. Biol. Chem. 261, 4889-4894]. In this study, we compare the amounts of CML and FL in normal human lens proteins, aged 0-79 years, using specific and sensitive assays based on selected ion monitoring gas chromatography-mass spectrometry. Our results indicate that the lens content of FL increases significantly between infancy and about age 5 but that there is only a slight, statistically insignificant increase in FL between age 5 and 80 (mean +/- SD = 1.4 +/- 0.4 mmol of FL/mol of Lys). In contrast, the lens content of the oxidation product, CML, increased linearly with age, ranging from trace levels at infancy up to 8 mmol of CML/mol of lysine at age 79. The ratio of CML to FL also increased linearly from 0.5 to 5 mol of CML/mol of FL between age 1 and 79, respectively. These results indicate that CML, rather than FL, is the major product of glycation detectable in adult human lens protein. The age-dependent accumulation of CML in lens protein indicates that products of both glycation and oxidation accumulate in the lens with age, while the constant rate of accumulation of CML in lens with age argues against an age-dependent decline in free radical defense mechanisms in this tissue.     

Enhancement of the horseradish peroxidase-catalyzed chemiluminescent oxidation of cyclic diacyl hydrazides by 6-hydroxybenzothiazoles

6-Hydroxybenzothiazole, 2-cyano-6-hydroxybenzothiazole, and 2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid (dehydroluciferin) dramatically enhance light emission from the horseradish peroxidase conjugate catalyzed oxidation of luminol, isoluminol, N-(6-aminobutyl)-N-ethyl isoluminol, and 7-dimethylaminonaphthalene-1,2-dicarboxylic acid hydrazide by either peroxide or perborate. Light emission is enhanced by up to 1000-fold, which is an improvement over the enhancement previously observed using firefly luciferin (4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid). Enhancement is influenced by enhancer concentration and pH. Spectral scans of light emitted in enhanced and unenhanced reactions are similar, suggesting that aminophthalate products, and not the enhancers, are the emitters.     

Mechanistic studies with general acyl-CoA dehydrogenase and butyryl-CoA dehydrogenase: evidence for the transfer of the beta-hydrogen to the flavin N(5)-position as a hydride

Butyryl-CoA dehydrogenase from Megasphera elsdenii catalyzes the exchange of the alpha- and beta-hydrogens of substrate with solvent [Gomes, B., Fendrich, G., & Abeles, R. H. (1981) Biochemistry 20, 1481-1490]. The stoichiometry of this exchange was determined by using 3H2O label as 1.94 +/- 0.1 per substrate molecule. The rate of 3H label incorporation into substrate under anaerobic conditions is monophasic, indicating that both the alpha- and beta-hydrogens exchange at the same rate. The exchange in 2H2O leads to incorporation of one 2H each into the alpha- and the beta-positions of butyryl-CoA, as determined by companion 1H NMR experiments and confirmed by mass spectroscopic analysis. In contrast, with general acyl-CoA dehydrogenase from pig kidney, only exchange of the alpha-hydrogen was found. The beta-hydrogen is the one that is transferred (reversibly) to the flavin 5-position during substrate dehydrogenation. This was demonstrated by reacting 5-3H- and 5-2H-reduced 5-deaza-FAD-general acyl-CoA dehydrogenase with crotonyl-CoA. Only one face of the reduced flavin analogue is capable of transferring hydrogen to substrate. The rate of this reaction is 11.1 s-1 for 5-deaza-FAD-enzyme and 2.2 s-1 for [5-2H]deaza-FAD-enzyme, yielding an isotope effect of 5. These values compare with a rate of 2.6 s-1 for the reaction of native reduced enzyme with crotonyl-CoA. The two reduced enzymes (normal vs. 5-deaza-FAD-enzyme) thus react at similar rates, indicating a similar mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elucidation of the antigenic determinant in lipotropin for two monoclonal antibodies

Two monoclonal antibodies were found to give enhanced affinity for β-lipotropin when mixed, as evidenced by competitive radioimmunoassay. Both monoclonals were found to react with a pentapeptide Ala-Glu-Leu-Glu-Tyr, which is a sequence of high local hydrophilicity within the N-terminal section of β-lipotropin.     

Daily ration of adult perch, Perca fluviatilis L. during summer in Loch Leven, Scotland

Adult perch, Perca fluviatilis L., were sampled at 3-hourly intervals throughout 24-h periods from June-September 1971, and in June and August 1972. The wet weight of each major food component from the stomachs was expressed in parts per 10 000 of the fresh weight of the fish. From samples at capture, and others taken from caged fish at known time-intervals after capture, the rate of stomach evacuation was estimated. Food consumption between netting times was calculated as the increment between successive initial values of stomach contents, plus the amount evacuated in the interval. As the evacuation rate of caged fish was slower than that of free fish the estimates of food turnover are minimal ones. The perch were consuming 6.5 % of their wet body weight per day in June, and this ration had decreased to 3.2% by September.     

Factors affecting recurrent shoot multiplication in in vitro cultures of 17- to 20-year-old douglas fir trees

Summary The effects of sucrose concentration, addition of ammonium nitrate, and exposure to N⁶-benzyl-adenine (BA) on multiplication potential with shoots derived from shoot cultures of 17- to 20-yr-old Douglas fir trees [Pseudotsuga menziesii (Mirb.) Franco] were compared. Each of these conditions, when compared independently, affected recurrent shoot multiplication and influenced shoot development, as measured by the abundance of shoot apices. Sucrose concentration was influential, the use of 25 g · liter⁻¹ providing twice the multiplication obtained with 20 g · liter⁻¹, and 14 × that obtained with the 30 g · liter⁻¹ concentration routinely used (tree 11). Ammonium nitrate usage also improved multiplication, a 2.5 times improvement being obtained after incorporation of 100 mg · liter⁻¹ NH₄NO₃ into the medium (tree 33). Shoot cultures were responsive but relatively sensitive to addition of BA, the best improvement in multiplication (5 times) being obtained with brief exposures to 3 mg · liter⁻¹ BA (tree 11). Although shoot cultures were responsive to the conditions investigated, differences in shoot multiplication and development were not displayed for several weeks. It was not possible therefore to repeat all the treatments with more than one genotype; however, when this was possible a genotype-dependent variation in response was evident.     

Tyrosine aminotransferase deficiency in mink (Mustela vision): A model for human tyrosinemia II

Mink pseudodistemper, a recessive disease associated with high blood tyrosinelevels, is an animal analogue of the human inborn error of metabolism, tyrosinemia II. Affected mink and man have eye and skin lesions. Affected mink have no hepatic tyrosine aminotransferase (TAT) activity, as measured immunologically and biochemically. Hepatic mitochondrial aspartate aminotransferase is increased to 188% of control. This new genetic animal model of TAT deficiency should allow new studies of tyrosine metabolism.Supported by NIH Grants AM-17253 (LAG), 5T32-AM-07093 (LAG), and RCDA AM-0008 (LAG), grants from the Wisconsin State Mink Advisory Board, and a BRSG grant for the Graduate School of the University of Wisconsin, Madison, Publication No. 82 of the dermatology research laboratories of Duke University Medical Center.