Ferritin-iron increases killing of Chinese hamster ovary cells by X-irradiation

Abstract. Stationary-phase Chinese hamster ovary cells were cultured in medium containing ferritin (-19% iron by weight) added at concentrations ranging from 0 to 128 μ g/ml. One set of cultures was unirradiated, and another set was exposed to 4.0 Gy of X-ray. Clonogenic cell survival was assessed in each set of cultures. In the absence of added ferritin, 4.0 Gy killed approximately 50% of the cells. In the absence of radiation, ferritin was not toxic at less than 48 μ g/ml; above 48 μ g/ml, toxicity increased with concentration. Apoferritin was not toxic at any concentration tested (up to 1000 μ g/ml). Although 32 μg/ ml ferritin, reflecting only a 3–6 fold increase in iron concentration over normal serum, was not toxic, it reduced the survival of X-irradiated cells by an additional 75%. These results indicate that a sublethal concentration of ferritin can be a potent radiosensitizer. This suggests the possibility that high body iron stores may increase susceptibility to radiation injury in humans.     

Radiation inactivation probe of membrane-bound enzymes: gamma-glutamyltranspeptidase, aminopeptidase N, and sucrase

gamma-Glutamyltranspeptidase (GGT), aminopeptidase N (AP-N), and sucrase in purified rabbit intestinal brush border membrane vesicles were irradiated in situ at -135 degrees C using high energy electrons. Surviving activities of the enzymes were measured as a function of radiation dose, and the functional unit target sizes (corresponding to carbohydrate-free polypeptides) were determined using target analysis. The in situ functional unit sizes were GGT 59 kDa, AP-N 59 kDa, and sucrase 63 kDa. Together with biochemical data determined previously, it is concluded that the noncovalently attached large (approximately 40 kDa) and small (approximately 25 kDa) subunits of GGT are both required for catalytic activity. Furthermore, these data suggest that (i) the membrane-bound form of AP-N consists of one or more noncovalently attached subunits of 59 kDa, each of which is enzymatically active; and (ii) in situ sucrase activity is associated with a subunit of 63 kDa which is noncovalently attached within the sucrase-isomaltase complex.     

Relationships between liquid- and solid-phase antibody association characteristics: implications for the use of competitive ELISA techniques to map the spatial location of idiotopes

A liquid-phase assay system based on small-zone size-exclusion chromatography was used to examine the binding of a monoclonal anti-idiotopic antibody, F6, to its idiotope on the murine plasmacytoma IgA, TEPC-15. Chromatographic behavior revealed a strong association between T-15 and F6, which was previously characterized by solid-phase immunoassay as recognizing a nonbinding site epitope of the T-15. This chromatographic pattern suggests that the inability of the hapten phosphorylcholine to inhibit the anti-idiotope:idiotope relationship in solid-phase immunoassay might be equally explained by the low affinity of the hapten relative to the high affinity of the anti-idiotope antibody. Bivalent interactions between solid-phase IgA and liquid-phase IgG should enhance the binding of the anti-idiotope to an extent that would prevent the hapten from dissociating the complex. Under these solid-phase assay conditions, observation of hapten inhibition may, in some cases, indicate site specificity, but absence of inhibition cannot be interpreted. Computer simulations of solid-phase hapten inhibition scenarios were used to evaluate the qualitative nature of binding inhibition profiles expected under various conditions of liquid- and solid-phase reactant affinities and concentrations. The apparently unusual inhibition curves previously observed in the T-15:anti-T-15 studies in which the degree of binding inhibition by hapten appeared to be independent of anti-idiotope concentration may be predictable in cases of excess solid-phase epitope; the plateau inhibition value is a function of relative affinity constants and concentrations of solid-phase and inhibitor components. The results additionally suggest that the affinity of a liquid-phase antibody may modulate the effective concentration of solid-phase epitope.     

Non-cross-reactive monoclonal antibodies to human chorionic gonadotropin generated after immunization with a synthetic peptide

Noncross-reactive monoclonal antibodies specific for human chorionic gonadotropin (hCG) were obtained after pre-selection for submolecular specificity with a synthetic peptide immunogen. Mice were immunized with a synthetic peptide representing a segment unique to the beta-subunit of hCG (amino acid residues 109-145), conjugated to diphtheria toxoid. We then derived nine different hybridomas that secreted monoclonal antibodies reactive with both native hCG and isolated C-terminal peptide, after somatic cell hybridization of immune spleen cells with a nonsecretory myeloma cell line. None of the nine monoclonal antibodies, termed beta-hCG-CTPa1----a9, reacted with hLH, hFSH, or hTSH, although these pituitary hormones display extensive amino acid sequence homology with hCG. The noncross-reactive anti-beta-hCG monoclonal antibodies show apparent association constants on the order of 10(9) to 10(10) M-1. A sandwich-type enzyme-linked immunosorbent assay was set up with cut-off values of around 5 mIU/ml. These antibodies might have important implications for: a) improving the diagnosis and clinical management of pregnancy; b) monitoring the course of development of carcinomas which secrete the hormone, through in vitro assays or in vivo radioimmunodetection; c) evaluating the antibodies' therapeutic potential against such carcinomas; d) studying the biologic functions of the C-terminal segment of beta-hCG; and e) addressing the anti-fertility effect of antibodies raised against that segment.     

Formation of an infinite beta-sheet arrangement dominates the crystallization behavior of lambda-type antibody light chains

The packing interactions in crystals of human lambda-type antibody light chain dimers have been reviewed. These homologous proteins are composed of individually specific variable domains, but all have very similar constant domain sequences. The proteins do not emulate each other in their overall crystallization behavior: each attains an individually characteristic space group or unit cell dimensions. However, each of these protein crystals has one unit cell dimension in common, 72.4(+/- 0.2) A. Examination of the protein packing in these crystals reveals that the common cell dimension is a consequence of a packing arrangement of their constant domains, which is conserved in all three crystals. In this striking arrangement, beta-sheets of adjacent constant domains are placed in juxta-position to form an "infinite chain". Although this constant domain packing pattern is rigorously conserved, the variable domain packing arrangements in each of these crystals are different. The conservation of the "infinite" beta-sheet pattern suggests that the constant domain interactions dominate the thermodynamic energy of lattice formation, probably through a combination of specific hydrogen bond formations and by a decrease in the solvent-accessible surface. A single amino acid substitution prohibits this characteristic interneighbor hydrogen bond pattern in the homologous kappa-type light chains. This may explain the observation that very few kappa-type light chains have been crystallized.     

Age-related deficiency in the perceived strength of six odorants

A group of 20 elderly persons (70–89 yr) and a controlgroup of 20 young persons (18–25 yr) made magnitude estimationsof five concentration levels of six odorants and of five concentrationlevels of a tastant, NaCl. Relative to the estimations of thesalt solutions, the elderly's estimations of all six odorantswere lower than those of the young. This outcome substantiatesan earlier finding that, at least for one odorant (iso-amylbutyrate), old age blunts perceived odor strength more frequentlyand seriously than gustatory strength. The present experimentbroadens the picture and leads to the conclusion that age-relatedhyposmia is likely to affect the perception of many, if notall, odors. The six odorants were selected on the basis of structuraldiversity, hedonic tone, and earlier psychophysical study ofthem. They include three pleasant odors (iso-amyl butyrate,benzaldehyde, and d-limonene), one foul smelling (pyridine),and two relatively neutral ones (ethyl and iso-amyl alcohol).To a first approximation age-related weaknesses to these compoundscan be characterized as a constant per cent reduction of olfactorystrength across concentration level. tion level.     

Increased release of cyclic adenosine monophosphate into jugular vein in response to isoproterenol administration

Catecholamine administration elevates plasma cyclic AMP (cAMP) levels but the source of the cAMP is unknown. To determine possible sources, plasma cAMP levels were determined in blood vessels across the head, liver, kidney and lung in anesthetized dogs infused with the beta-adrenergic agonist, isoproterenol. Only the head showed an increased release of cAMP into the blood. The kidneys removed cAMP from the blood while liver and lung showed no change. This in vivo demonstration of release of cAMP from the head represents contributions from brain and facial muscles and may be a useful approach to study brain involvement in the action of various hormones and drugs.     

Enzymatic basis of macrophage activation. Kinetic analysis of superoxide production in lysates of resident and activated mouse peritoneal macrophages and granulocytes

To compare the kinetics of the O-2-generating enzyme in nonactivated and activated macrophages and granulocytes from the mouse peritoneal cavity, we sought conditions in which the activity of this enzyme in cell lysates was comparable to that in intact cells. Pretreatment of macrophages with 10 mM diethyldithiocarbamate inhibited endogenous superoxide dismutase by 70% and enhanced O-2 secretion up to 15-fold, so that it was comparable to H2O2 secretion. O-2 secretion was terminated by detergent lysis and reconstituted by addition of NAD(P)H to the lysates. Optimal detection of O-2 production in lysates depended on prior stimulation of the respiratory burst, lysis with 0.05% deoxycholate rather than any of 4 other detergents or sonication, acetylation of the cytochrome c used as an indicator, and addition of NADPH rather than NADH. Kinetic analysis using NADPH-reconstituted deoxycholate lysates, together with spectra of oxidized and reduced cells, failed to reveal either marked differences in the Vmax of the O-2-generating enzyme or correlations between O-2 secretion and cytochrome b559 content among 5 macrophage populations whose H2O2 secretion ranged from 0 to 365 nmol/90 min/mg of protein. In contrast, the Km of the oxidase for NADPH varied markedly and inversely with the capacity of the intact cells to secrete O-2 or H2O2: J774G8 histiocytoma cells, 1.43 mM; resident macrophages, 0.41 mM; proteose peptone-elicited macrophages, 0.20 mM; casein-activated macrophages, 0.05 mM; NaIO4-activated macrophages, 0.05 mM; and granulocytes, 0.04 mM. These results suggest that macrophage activation, a process that enhances oxygen-dependent antitumor and antimicrobial functions, may equip the cell to secrete increased amounts of reactive oxygen intermediates largely by increasing the affinity of the oxidase for NADPH.