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.     

[14C]-Glucose Metabolism during Shoot Bud Development in Cultured Cotyledon Explants of Pinus radiata

Excised cotyledons of Pinus radiata D. Don cultured under shoot-forming(plus benzyladenine) and non shoot-forming (minus benzyladenine)conditions for 10 and 21 days were fed U-[¹⁴C]-glucose for 3h in the light followed by a 3 h chase period. The labellingof individual metabolites as well as ¹⁴C incorporation intoprotein was assessed. It was found that the general metabolicpatterns were qualitatively the same in shoot-forming and nonshoot-forming conditions, however, metabolism leading to respirationas well as to the synthesis of some amino acids and proteinsynthesis was enhanced in the shoot-forming cultures. (Received February 16, 1987; Accepted July 8, 1987)     

Direct Evidence for Changes in the Resistance of Legume Root Nodules to O2 Diffusion

Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O₂ diffusion. Here we describeexperiments using O₂ specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O₂ concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m^–3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O₂ concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O₂ concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O₂ concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O₂ fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O₂ concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N₂or air. Key words: Oxygen, root nodules, air spaces     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Interaction of acyl coenzyme A substrates and analogues with pig kidney medium-chain acyl-coA dehydrogenase

Several alkylthio coenzyme A (CoA) derivatives (from ethyl- to hexadecyl-SCoA) have been synthesized to probe the substrate binding site in the flavoprotein medium-chain acyl-CoA dehydrogenase from pig kidney. All bind to apparently equivalent sites with a stoichiometry of four per tetramer. A plot of log Kd vs: hydrocarbon chain length is linear from 2 to 16 carbons with a free energy of binding of 390 cal/methylene group. These data suggest an acyl-binding site of moderate hydrophobicity and imply that the observed substrate specificity of the medium-chain dehydrogenase is not achieved simply by the length of the hydrocarbon binding pocket. Extrapolation of the graph to zero chain length predicts a Kd of 1 mM for the CoA moiety. The difference between this value and the experimentally determined value of 206 microM may be attributed to a contribution from the ionization of the sulfhydryl group in CoASH. The interaction of several eight-carbon intermediates of beta-oxidation (trans-2- and trans-3-octenoyl-CoA and L-3-hydroxy- and 3-ketooctanoyl-CoA) with the dehydrogenase has also been studied. All but the L-3-OH derivative bind tightly to the enzyme (with Kd values in the 50-90 nM range) and are very effective inhibitors of the dehydrogenation of octanoyl-CoA. The trans-3-enoyl analogue produces an immediate, intense, long-wavelength band (lambda max = 820 nm), which probably represents a charge-transfer interaction between the delocalized alpha-carbanion donor and oxidized flavin as the acceptor. The L-3-OH analogue is a reductant of the flavin, yielding 3-ketooctanoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The preparation of deglycosylated ricin by recombination of glycosidase-treated A- and B-chains: effects of deglycosylation on toxicity and in vivo distribution

Deglycosylation of ricin may be necessary to prevent the entrapment of antibody-ricin conjugates in vivo by cells of the reticuloendothelial system which have receptors that recognise the oligosaccharide side chains on the A- and B-chains of the toxin. Carbohydrate-deficient ricin was therefore prepared by recombining the A-chain, which had been treated with alpha-mannosidase, with the B-chain, which had been treated with endoglycosidase H or alpha-mannosidase or both. By recombining treated and untreated chains, a series of ricin preparations was made having different carbohydrate moieties. The removal of carbohydrate from the B-chain did not affect the ability of the toxin to agglutinate erythrocytes, and alpha-mannosidase treatment of the A-chain did not affect its ability to inactivate ribosomes. The toxicity of ricin to cells in culture was only reduced in those preparations containing B-chain that had been treated with alpha-mannosidase, when a 75% decrease in toxicity was observed. The toxicity of the combined ricin preparation to mice varied from double to half that of native ricin, depending on the chain(s) treated and the enzymes used. Removal of carbohydrate greatly reduced the hepatic clearance of the toxin and the levels of toxin in the blood were correspondingly higher. These results suggest that antibody-ricin conjugates prepared from deglycosylated ricin would be cleared more slowly by the liver, inflict less liver damage, and have greater opportunity to reach their target.     

Linkage analysis of chromosome 17 markers in British and South African families with neurofibromatosis type 1

Nine markers from the pericentromeric region of chromosome 17 were typed in 16 British and five South African families with neurofibromatosis type 1 (NF1). The markers--p17H8, pHHH202, and EW204--were linked to NF1 at recombination fractions less than 1%. No evidence of locus heterogeneity was detected. Inspection of recombinant events in families informative for several markers suggests that the NF1 gene is located between the markers EW301 (cen-p11.2) and EW206 (cen-q12) and possibly distal to pHHH202 (q11.2-q12).     

Inactivation of two-electron reduced medium chain acyl-CoA dehydrogenase by 2-octynoyl-CoA

The acetylenic thioester, 2-octynoyl-CoA, inactivates medium chain acyl-CoA dehydrogenase from pig kidney by two distinct pathways depending on the redox state of the FAD prosthetic group. Inactivation of the oxidized dehydrogenase occurs with labeling of an active site glutamate residue and elimination of CoASH. Incubation of the reduced dehydrogenase with 2-octynoyl-CoA rapidly forms a kinetically stable dihydroflavin species which is resistant to reoxidation using trans-2-octenoyl-CoA, molecular oxygen, or electron transferring flavoprotein. The reduced enzyme derivative shows extensive bleaching at 446 nm with shoulders at 320 and 380 nm. Denaturation of the reduced derivative in 80% methanol yields a mixture of products which was characterized by HPLC, by uv/vis, and by radiolabeling experiments. Approximately 20% of the flavin is recovered as oxidized FAD, about 40% is retained covalently attached to the protein, and the remainder is distributed between several species eluting after FAD on reverse-phase HPLC. The spectrum of one of these species ressembles that of a N(5)-C(4a) dihydroflavin adduct. These data suggest that a primary reduced flavin species undergoes various rearrangements during release from the protein. The possibility that the inactive modified enzyme represents a covalent adduct between 2-octynoyl-CoA and reduced flavin is discussed. Analogous experiments using enzyme substituted with 1,5-dihydro-5-deaza-FAD show rapid and quantitative reoxidation of the flavin by 0.5 eq of 2-octynoyl-CoA.     

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.