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.     

Novel antithrombotic therapy.

The prospects for more effective and safe antithrombotic therapy appear promising. I have presented only a portion of all novel antithrombotic agents currently under basic and clinical investigations. A discussion of additional new antithrombotic drugs is presented elsewhere.     

Electron paramagnetic resonance spectroscopy of lactoperoxidase complexes: clarification of hyperfine splitting for the NO adduct of lactoperoxidase

Electron paramagnetic resonance (EPR) studies of the nitrosyl adduct of ferrous lactoperoxidase (LPO) confirm that the fifth axial ligand in LPO is bound to the iron via a nitrogen atom. Complete reduction of the ferric LPO sample is required in order to observe the nine-line hyperfine splitting in the ferrous LPO/NO EPR spectrum. The ferrous LPO/NO complex does not exhibit a pH or buffer system dependence when examined by EPR. Interconversion of the ferrous LPO/NO complex and the ferric LPO/NO2- complex is achieved by addition of the appropriate oxidizing or reducing agent. Characterization of the low-spin LPO/NO2- complex by EPR and visible spectroscopy is reported. The pH dependence of the EPR spectra of ferric LPO and ferric LPO/CN- suggests that a high-spin anisotropic LPO complex is formed at high pH and an acid-alkaline transition of the protein conformation near the heme site does occur in LPO/CN-. The effect of tris(hydroxymethyl)aminomethane buffer on the LPO EPR spectrum is also examined.     

Hinging of rabbit myosin rod

The question of hinging in myosin rod from rabbit skeletal muscle has been reexamined. Elastic light scattering and optical rotation have been used to measure the radius of gyration and fraction helix, respectively, as a function of temperature for myosin rod, light meromyosin (LMM), and long subfragment 2 (long S-2). The radius of gyration vs temperature profile of myosin rod is shifted with respect to the optical rotation melting curve by about -5 degrees C. Similar studies on both LMM and long S-2 show virtually superimposable profiles. To correlate changes in the secondary structure with the overall conformation, plots of radius of gyration vs fraction helix are presented for each myosin subfragment. Myosin rod exhibits a marked decrease in the radius of gyration from 43 nm to approximately 35 nm, while the fraction helix remains at nearly 100%. LMM and long S-2 did not show this behavior. Rather, a decrease in the radius of gyration of these fragments occurred with comparable changes in fraction helix. These results are interpreted in terms of hinging of the myosin rod within the LMM/S-2 junction.     

Effect of selection intensity and population size on percent oil in maize,Zea mays L

Summary The effect of selection intensity and population size on the response to selection for percent oil in the grain of maize (Zea mays L.) was evaluated in a replicated experiment over ten cycles of selection. An open-pollinated variety, Armel's Reid Yellow Dent, was divided into subpopulations of 6,10 and 50 plants. Selection proportions of 17% and 5% were imposed upon each subpopulation. Selection was based on the percentage of oil in individual kernels as determined by wide-line nuclear magnetic resonance spectroscopy. As expected, total response to selection increased with larger population sizes and selection intensities. The concave shape of the response curves suggested that an appreciable part of the genetic variance can be attributed to additive genes at high initial frequencies, dominance genes at low initial frequencies, or to the generation of negative linkage disequilibrium due to selection. The consistently greater loss of vigor experienced by the more intensely selected populations reflects the enhancement of inbreeding due to artificial selection, an effect that increases with the intensity of selection. The results indicate that combined selection, based on kernels and using within- and amongfamily information, will be more efficient than other conventional selection procedures, including the normal combined scheme where selection is based on plants.Deceased     

Intercellular structure in a many-celled magnetotactic prokaryote

A many-called magnetotactic prokaryote obtained from brackish water was observed to possess intercellular connections at points of contact between the outer membranes of constituent cells. Each aggregate organism consisted of 10 to 30 individual Gram-negative cells containing material with the appearance of poly--hydroxybutyrate and magnetosomes of unusual arrangement, structure and composition. The aggregate, which possessed prokaryotic-type flagella arranged at the outwards surfaces of each cell, showed motility indicative of co-ordination between individual component cells. These results suggest that this organism could be a multicellular prokaryote.     

Immunoblot analysis of cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin in corpora lutea of cyclic and late-pregnant sheep

The specific contents of cytochrome P-450scc and adrenodoxin in corpora lutea of late pregnant sheep were, respectively, 1/5 and 1/8 that of corpora lutea of the oestrous cycle, suggesting lower steroidogenic enzyme capacity in the former. The contents of Complex V proteins were also lower in the corpora lutea of late pregnancy. It was observed in the immunoblots of both Complex V and cytochrome P-450scc that immunoreactive bands of molecular weights lower than the native proteins were present in the samples from corpora lutea of late pregnancy, indicative of degradation of the native enzymes. It is concluded that corpora lutea of sheep during late pregnancy have a much lower enzyme capacity for steroidogenesis than do those of the oestrous cycle (mid-luteal phase) due to a reduction in the content of cytochrome P-450scc and adrenodoxin. The reduction in the levels of steroidogenic enzyme proteins appears to be unspecific and probably reflects an overall demise in mitochondrial functions.     

Cultivar differences in the rate of nitrate uptake by intact wheat plants as related to growth rate

Six Argentinian wheat ( Triticum aestivum L.) cultivars grown in nutrient solutions in controlled environment were compared for their nitrate uptake rates on a root dry weight basis. Up to 3-fold differences were observed among the cultivars at 16, 20 and 24 days from germination, either when measured by depletion from the nutrient solution in short-term experiments, or by total N accumulation in the tissue during 8 days.
No differences in total N concentration in root or shoots were found among cultivars. Although the different cultivars showed significant differences in shoot/root ratio and nitrate reductase activity (EC 1.6.6.1) in the roots, none of these parameters was correlated with the nitrate uptake rate. However, nitrate uptake was found to be positively correlated (r = 0.99) with the shoot relative growth rate of the cultivars. The three cultivars with the highest nitrate uptake rates and relative growth rates showed a positive correlation between root nitrate concentration and uptake. However, this correlation was not found in the cultivars with the lowest growth and uptake rates.
Our results indicate that the difference in nitrate uptake rate among these cultivars may only be a consequence of their differences in growth rate, and it is suggested that at least two mechanisms regulate nitrate uptake, one working when plant demand is low and another when plant demand is high.     

Localization of follicle regulatory protein in the porcine ovary

The granulosa cell secretes a protein (follicle regulatory protein: FRP) that affects the responsiveness of other follicles to gonadotropin stimulation. This protein was purified, partially characterized, and rabbit antisera as well as monoclonal antibodies were prepared against FRP. Fixed sections of porcine ovaries were prepared on slides and then incubated with the monoclonal antibody or polyclonal antisera and then incubated with either biotinylated mouse IgM or rabbit IgG antisera, respectively. These sections were then incubated with avidin conjugated to horseradish peroxidase, followed by substrate. Staining with both the monoclonal antibody and the antisera was present in the cytoplasm of granulosa cells of small- or medium-sized antral follicles. Staining distribution was localized preferentially to cells near the basal lamina; the antral granulosa cells of viable follicles did not stain. Neither primordial follicles nor pre-antral follicles (less than 300 microns in diameter) showed any positive staining. Thecal cells were not stained in follicles less than 5 mm in diameter, whereas some large follicles (greater than 5 mm) contained staining in the theca. In the latter, specific granulosa staining was only weakly positive with the polyclonal antibody and negative with the monoclonal antibody. Atretic follicles contained significant staining of all epithelial cells adjacent to the basal lamina by both the monoclonal and polyclonal antibody preparations. Staining of the luteal ovary by the monoclonal antibody was limited to the large luteal cells. These findings suggest that FRP is produced by the granulosa cells of porcine follicles at the stage of maturation corresponding to 0.5 mm in diameter. As the viable follicle increases in size, production of FRP in the granulosa is reduced below the detectable level when the follicle exceeds 5 mm in diameter. The main source of FRP during the luteal phase is the large cell of the corpus luteum.     

Effect of glycerol on thermolysin-catalyzed peptide bond synthesis

The effect of glycerol on the hydrolytic activity of thermolysin (EC 3.4.24.4) has been compared with the effect on the condensation of N-benzyloxycarbonyl-L-aspartic acid with L-phenylalanine methyl ester to form N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (Z X Asp X Phe X OMe), the precursor to the sweet-tasting compound L-aspartyl-L-phenylalanine methyl ester. Hydrolytic activity was measured by the degradation of azocasein and furylacryloyl-L-glycyl-L-leucinamide. Increasing concentrations of glycerol reversibly inhibited the hydrolytic activity of the enzyme toward both substrates. The inclusion of glycerol in the synthetic medium facilitated the production of Z X Asp X Phe X OMe in a water-soluble system but reduced the initial rate of peptide synthesis. Glycerol stabilized thermolysin against thermal denaturation.